Appl Microbiol Biotechnol (2007) 73:1251–1258
DOI 10.1007/s00253-006-0718-6
MINI-REVIEW
Applications of whole-cell bacterial sensors in biotechnology
and environmental science
Kiyohito Yagi
Received: 14 August 2006 / Revised: 30 September 2006 / Accepted: 12 October 2006 / Published online: 17 November 2006
# Springer-Verlag 2006
Abstract Biosensors have major advantages over chemical
or physical analyses with regard to specificity, sensitivity,
and portability. Recently, many types of whole-cell bacte-
rial biosensors have been developed using recombinant
DNA technology. The bacteria are genetically engineered to
respond to the presence of chemicals or physiological
stresses by synthesizing a reporter protein, such as
luciferase, β-galactosidase, or green fluorescent protein. In
addition to an overview of conventional biosensors, this
minireview discusses a novel type of biosensor using a
photosynthetic bacterium as the sensor strain and the crtA
gene, which is responsible for carotenoid synthesis, as the
reporter. Since bacteria possess a wide variety of
stress-response mechanisms, including antioxidation, heat-
shock responses, nutrient-starvation, and membrane-dam-
age responses, DNA response elements for several
stress-response proteins can be fused with various reporter
genes to construct a versatile set of bacterial biosensors for
a variety of analytes. Portable biosensors for on-site
monitoring have been developed using a freeze-dried
biosensing strain, and cell array biosensors have been
designed for high-throughput analysis. Moreover, in the
future, the use of single-cell biosensors will permit detailed
analyses of samples. Signals from such sensors could be
detected with digital imaging, epifluorescence microscopy,
and/or flow cytometry.
Keywords Whole-cell biosensor . Reporter gene .
Luciferase . β-galactosidase . Green fluorescent protein . crtA
K. Yagi (*)
Graduate School of Pharmaceutical Sciences,
Osaka University,
1-6 Yamada-oka,
Suita, Osaka 565-0871, Japan
e-mail: yagi@phs.osaka-u.ac.jp
Introduction
Biosensors are devices that make use of a biological
component to obtain information. They can be designed
to detect small amounts of a chemical, or to report
physiological and biochemical characteristics of a sample.
The major advantages of using biological components in
sensors are their good specificity, sensitivity, and portabil-
ity. Moreover, unlike chemical or physical analyses,
biological systems do not require large and expensive
instruments. So far, enzymes, antibodies, subcellular com-
ponents, and microorganisms have mainly been used as the
biological components of the sensors. Although purified
enzymes are frequently used in biosensors due to their high
specificity, the purification procedure is laborious, expen-
sive, and time-consuming. The unstable nature of enzymes
has hampered the broad application of enzyme-based
biosensors to the fields of biotechnology and environmental
sciences. Microorganisms have several advantages over
purified enzymes as the biological component of biosen-
sors. Numerous kinds of microorganisms exist in the
natural environment and are maintained in culture collec-
tions, allowing the choice of a suitable strain for a given
purpose. Microorganisms, especially bacteria, proliferate
very rapidly under aerobic or anaerobic conditions in
relatively inexpensive media. Another important advantage
of a whole-cell biosensor is the ability to analyze the
sample through processes in which many enzymes are
involved, such as the respiratory system and fermentation.
Methods for immobilizing bacteria in a membrane or gel-
matrix have been developed to construct bacterial biosen-
sors. The physiological response of immobilized bacteria to
an analyte can be transmitted to a sensing device, such as
an oxygen electrode, to monitor organic compounds in the
sample. Several reviews of bacterial biosensors have been
published (D’Souza 2001; Matrubutham and Sayler 1998;
1252 Appl Microbiol Biotechnol (2007) 73:1251–1258

Nomura and Karube 1996; Riedel 1998). Recently, a novel Table 1 Characteristics of commonly used reporter genes for
type of bacterial biosensor was developed using recombi- bacterial biosensors
nant DNA technology (Fig. 1). The bacteria are genetically Gene Protein Advantages Defects
engineered to respond to the presence of chemicals or
physiological stresses through the synthesis of a reporter lux Bacterial luciferase Ease of Heat lability
protein, such as luciferase, β-galactosidase (β-gal), or green measurement
Rapid response Oxygen requirement
fluorescent protein (GFP). This review focuses on the
luc Firefly luciferase High sensitivity Exogenous substrate
construction of recombinant bacterial biosensors and their requirement
applications in biotechnology and environmental sciences. No heat lability Oxygen requirement
gfp Green fluorescent Autofluorescence Low sensitivity
protein (jelly fish) No substrate Lag time before
Reporter genes requirement expression
lacZ β-gal (E. coli) Detection by Exogenous substrate
naked eye requirement
Many attempts have been made to use reporter genes fused
Wide variety of Complicated
with a DNA response element (RE) for an analyte or
detection colorimetric
molecules induced by physiological stress. Table 1 summa- methods operation
rizes the characteristics of commonly used reporter genes.
King et al. first reported the use of the bacterial luciferase
(lux) gene to construct a bacterial biosensor for the 2004; Heitzer et al. 1994; Stocker et al. 2003; Selifonova et
detection of naphthalene (King et al. 1990). Bacterial al. 1993). Similarly, the firefly luciferase (luc) gene has
luciferase catalyzes the oxidation of long-chain fatty been used as the reporter gene (Kobatake et al. 1995;
aldehydes and reduced flavin mononucleotides to form the Tauriainen et al. 1998, 1999). Luc catalyzes the oxidation
corresponding fatty acid and FMN in the presence of of benzothiazole-thiazole luciferin to form bioluminescent
oxygen. This reaction produces bioluminescence. The oxyluciferin in the presence of O2 and ATP. Although Luc
catalytic subunits, α and β, are encoded by the luxA and has a higher sensitivity than Lux (Billard and DuBow
luxB genes, respectively, which are located in the lux 1998), the addition of the substrate and ATP is required for
operon. The lux operon consists of five genes, luxC, D, A, it to generate bioluminescence. On the other hand, Lux is
B, and E, and the expression of C, D, and E is required to heat-labile, and it is difficult to use in mammalian cells
form fatty acid reductases that synthesize and recycle fatty (Naylor 1999).
aldehydes (Billard and DuBow 1998; Wilson and Hastings GFP is a reporter whose autofluorescence can be
1998). To make a whole-cell biosensor, the lux operon was produced without the addition of an exogenous substrate
fused with a DNA RE for heavy metals and halogenated or ATP (Stiner and Halverson 2002; Shetty et al. 1999).
compounds, and the construct was introduced into Escher- The autofluorescence of GFP derives from the presence of a
ichia coli (Hay et al. 2000; Sticher et al. 1997; Werlen et al. covalently bound imidazolinone chromophore inside the
protein. The GFP gene was cloned from the jellyfish
Aequorea victoria in 1992 (Prasher et al. 1992), and mutant
gfp genes were generated to improve the fluorescent
properties of the wild-type protein (Misteli and Spector
1997; Welsh and Kay 1997). The mutant proteins had
increased stability (Siemering et al. 1996), altered spectral
properties (Heim and Tsien 1996), or improved signal
intensity (Crameri et al. 1996). Since then, GFP has been
used extensively to measure gene expression, identify
transplanted cells, and analyze differentiation processes in
mammalian systems. However, the application of GFP to
bacterial biosensors is limited for several reasons. It takes
time to form the active fluorophore after the synthesis of the
core protein, thus delaying the full fluorescence activity.
Fig. 1 Whole-cell bacterial biosensors made using recombinant DNA The presence of intrinsic fluorescence in bacterial cells also
technology. Bacteria receive extracellular signals through a receptor- limits the usefulness of this reporter, because of the high
dependent or independent pathway, and the signal-mediated activation
background signal.
of the response element (RE) induces the transcription of the reporter
gene. The reporter protein exhibits specific luminescence, fluores- β-gal is a well-known bacterial enzyme that is very
cence, or color development as the detectable signal widely used in molecular biology to monitor the transfec-
Appl Microbiol Biotechnol (2007) 73:1251–1258
tion efficiency of plasmid and virus vectors. Several
methods to detect β-gal activity have been developed using
a variety of substrates. The substrate most commonly used
for the colorimetric detection of β-gal is O-nitrophenyl β-
D-galactopyranoside. Although this colorimetric assay can
be done simply and rapidly, luminescence-based and
electrochemical assays with much higher sensitivity that
use 1,2-dioxetane substrates and p-aminophenyl-β-galacto-
pyranoside, respectively, have also been developed (Jain
and Magrath 1991; Biran et al. 1999). Bacterial biosensors
employing an inducible promoter and the β-gal gene have
been constructed to detect heavy metals (Scott et al. 1997),
halogenated compounds (Guan et al. 2000), and viruses
(Olivo et al. 1998). In particular, an electrochemical assay
of β-gal activity allows the continuous, in situ monitoring
of chemicals (Biran et al. 2000).
Novel reporter gene
In a recent study, my colleagues and I developed a novel
type of bacterial biosensor using a photosynthetic bacteri-
um, Rhodovulum sulfidophilum, for the sensor strain and
the crtA gene, which is responsible for carotenoid synthesis
(Fig. 2), as the reporter, to detect environmental pollutants
(Maeda et al. 2006; Fujimoto et al. 2006). This biosensor
indicates the presence of a pollutant by a bacterial color
change, without the need to add a special reagent or
substrate for color development. Moreover, no equipment
for detecting fluorescence or chemiluminescence under
light-shielded conditions is necessary to detect a pollutant,
which can be monitored in samples by the naked eye under
sunlight, even in areas where electricity is not available.
Fig. 2 Carotenoid synthesis pathway in the photosynthetic bacterium
Rhodovulum sulfidophilum
1253
Rhodovulum sulfidophilum belongs to a group of purple
photosynthetic bacteria that synthesize carotenoids through
the spheroiden pathway (Takaichi 2001; Takaichi et al.
2001). In this pathway, spheroidene (SE) is formed by O-
methylation of the C-1 hydroxy group of demethylspher-
oidene by O-methyltransferase (CrtF), and spheroidenone
(SO) is formed by ketolation at the C-2 of SE monoox-
ygenase (CrtA). SO is the predominant carotenoid under
semiaerobic conditions. The CrtA in these bacteria is
responsible for changing the color of the cultures from
yellow, due to SE, to red, due to SO (Yeliseev et al. 1996).
In a previous study, my colleagues and I isolated a CrtA-
deleted mutant of R. sulfidophilum (CDM2) that was made
by inserting a kanamycin cassette into the crtA gene; this
mutant accumulates SE and therefore displays a yellow
color in culture (Maeda et al. 2005). A sensor plasmid for
arsenite detection was constructed by fusing the crtA gene
and the arsenite DNA RE, and introducing the plasmid into
the CDM2 strain (Fujimoto et al. 2006). The color change
from yellow to red could be clearly recognized with the
naked eye at 5 μg/l arsenite.
Target analytes
Heavy metals
Microorganisms have a wide variety of defense mecha-
nisms against metals that exist in the natural environment.
Many attempts have been made to construct bacterial
biosensors that can detect small amounts of metals by
fusing metal DNA REs and/or regulatory protein genes
with various reporter genes. Biosensors using the DNA
region responsible for mercury resistance have been
reported (Condee and Summers 1992; Selifonova et al.
1993; Hansen and Sorensen 2000). MerR is a metallo-
regulatory protein that binds to Hg(II) and activates the
transcription of the mercury-resistance genes merTPCAD in
E. coli. The promoter region of the mer operon was fused
with a reporter gene, such as luxAB, to construct sensor
plasmids that respond to the presence of Hg(II). The
heterologous MerR binds to Hg(II) and activates the mer
promoter upstream of luxAB. Similarly, bacterial biosensors
that detect arsenite have been constructed by fusing reporter
genes with the promoter region of the ars operon (Corbisier
et al. 1993; Cai and DuBow 1997; Ramanathan et al. 1997;
Scott et al. 1997; Tauriainen et al. 1997; Fujimoto et al.
2006). The arsenite-resistance mechanism has been exten-
sively analyzed in E. coli. The ars operon, consisting of
two regulatory genes (arsR and arsD) and three structural
genes (arsA, arsB, and arsC), is responsible for arsenite
resistance (Wu and Rosen 1991; Kaur and Rosen 1992).
ArsR is a repressor that binds to the operator/promoter
1254
region of the ars operon (O/Pars) and prevents transcrip-
tion of the downstream genes for arsenite resistance. When
arsenite is present, ArsR binds with it, which causes ArsR
to undergo a conformational change and dissociate from the
operator (Shi et al. 1996). My colleagues and I constructed
an arsenite biosensor plasmid by fusing O/Pars, arsR, and
crtA, and introduced these constructs into the yellow-
colored CDM2 strain (Fig. 3). In the absence of arsenite,
ArsR binds to O/Pars to suppress the transcription of
downstream genes, maintaining the yellow cell color. When
arsenite binds to ArsR, the complex dissociates from the O/
Pars region to activate the transcription of the crtA gene,
resulting in the production of the red carotenoid SO.
Besides mercury and arsenite, biosensors to detect other
toxic metals have been constructed using gene-fusion
technology. Cadmium biosensors were constructed in
Staphylococcus aureus using lux (Corbisier et al. 1993)
and luc (Tauriainen et al. 1998). These biosensors also
responded to lead and antimony. A chromate biosensor was
constructed in Alcaligenes eutrophus using β-gal (Peitzsch
et al. 1998). A nickel biosensor was constructed in Ralstnia
eutropha using lux, to detect bioavailable nickel in soil
(Tibazarwa et al. 2001).
Fig. 3 Arsenite bacterial bio-
sensor using crtA as the reporter
gene
Appl Microbiol Biotechnol (2007) 73:1251–1258
DNA damage and various stresses
Bacterial biosensors that can detect genotoxicity more easily
and more rapidly than the Ames test have been developed
(Cheng Vollmer and Van Dyk 2004). When DNA is damaged
in E. coli, the RecA protein binds to single-stranded DNA to
form a nucleoprotein filament. This complex stimulates the
autocatalytic cleavage of LexA, which acts to repress SOS
genes including uvrA, uvrB, and recA, resulting in DNA
repair. To make a genotoxin biosensor, the recA gene, whose
transcription is regulated by the LexA protein, was fused
with luxCDABE (Vollmer et al. 1997) or lacZ (Nunoshiba
and Nishioka 1991) and introduced into E. coli. When DNA
damage increases in the presence of genotoxins, the recA
promoter is activated by the autodegradation of endogenous
LexA, resulting in the transcription of the reporter gene.
Biosensors have also been made with DNA REs for
stress-induced proteins, and are reviewed more extensively
elsewhere (Cheng Vollmer and Van Dyk 2004). In brief, the
heat-shock response was used to construct a sensor by
fusing luxCDABE to the promoter region of heat-shock
genes, such as dnaK (Hsp70) or grpE (Van Dyk et al. 1994),
which are involved in the Hsp70 molecular chaperone
Appl Microbiol Biotechnol (2007) 73:1251–1258
system. In this system, expression of the tagged dnaK or
grpE is induced following stress by the endogenous
upregulation of σ32, which mediates the E. coli heat-shock
response (Yura and Nakahigashi 1999). This sensor
monitors environmental pollution by detecting intracellular
protein damage. In a different biosensor, the oxidative
stress response is used to report the generation of hydrogen
peroxide. The catalase-encoding katG gene was fused with
luxCDABE and introduced into E. coli (Belkin et al. 1996).
When the endogenous OxyR protein, acting as a molecular
code for redox-related signaling (Kim et al. 2002), is
oxidized to form a disulfide bond, the oxidized protein
undergoes a conformational change to become a transcrip-
tional activator for the katG promoter. Superoxide dismut-
ase-encoding sodA gene has also been used to construct a
sensor for oxidative stress in E. coli (Lee and Gu 2003).
The endogenous SoxRS protein acts as a transcriptional
activator for the sodA promoter (Wu and Weiss 1992).
Biosensors responding to oxidative stress could be used to
evaluate the antioxidation effects of diet.
Since bacteria have a wide variety of stress-response
mechanisms other than antioxidation and heat-shock
responses, including nutrient-starvation and membrane-
damage responses, DNA REs and/or regulatory protein
genes for several stress-response proteins can be fused with
various reporter genes to construct a versatile set of
bacterial biosensors for a variety of analytes.
Characteristics of reporter genes
Stocker et al. constructed arsenite biosensors using lux, gfp,
or β-gal as the reporter gene, and compared their detection
limit and linearity range (Stocker et al. 2003). A good linear
response was obtained from 0–0.5 μM arsenate in the lux-
based biosensor. The lowest measurable concentration was
0.05 μM (3.9 μg/l). For the gfp-based biosensor, the rate of
GFP fluorescence increase could be approximated by linear
interpolation. The approximate linear range was found
between 0.1 and 0.6 μM. The sensitivity of the gfp-based
biosensor to arsenite was less than that of the lux-based
biosensor, with a lowest measurable concentration of
0.1 μM (7.8 μg/l). The β-gal-based biosensor was used in
a colorimetric qualitative paper-strip test for field applica-
tion. It could be used to determine whether a sample
contains arsenite at or below the current drinking water
limits of 10 μg/l. In a recent study, my colleagues and I
developed a crtA-based qualitative arsenite biosensor for
on-site monitoring (Fujimoto et al. 2006). The color change
could be clearly recognized with the naked eye at 5 μg/l in
liquid culture. For highly accurate and quantitative assays,
lux or gfp can be used as the reporter gene, and for simple
detection in the field, β-gal and crtA are appropriate.
1255
Portable biosensors for on-site monitoring
One important characteristic for a biosensor intended to
monitor environmental pollution is portability to permit on-
site monitoring. Gu et al. examined the optimal conditions
of freeze-drying for a recombinant bioluminescent bacteri-
um to maintain its viability and function during storage and
transport to a monitoring site (Gu et al. 2001). A portable
biosensor kit consisting of three parts, a freeze-dried
biosensing strain, a compact light-proof chamber, and a
luminometer, has since been constructed (Choi and Gu
2002). An even more simplified sensor was developed
using a freeze-dried recombinant bacterium for the on-site
detection of phenolic compounds by a color change (Shin et
al. 2005). For this biosensor, a sensor plasmid was
constructed by fusing the β-gal gene and the CapR
promoter, which is activated in the presence of phenol
(Park et al. 2003). The E. coli strain bearing the sensor
plasmid was freeze-dried and stored until use. The sensor
strain was then rehydrated, and X-galactopyranoside, a β-
gal substrate, was added to mediate the color change. As
described above, my colleagues and I also developed a
simplified arsenite biosensor using the crtA gene as the
reporter and photosynthetic bacteria as the host strain
(Fujimoto et al. 2006). The freeze-dried biosensor strain
retained its function after rehydration. Since the presence of
an analyte can be monitored by the naked eye without the
addition of a substrate for color development in this system,
it would also be appropriate for a portable biosensor kit for
on-site monitoring.
Cell array biosensors
Since stabilization and reusability are important factors for
a bacterial biosensor to be of practical use, many attempts
have been made to immobilize the sensor strain within
certain matrices. Agar has been used to immobilize a
recombinant sensor strain, and the response stability was
improved, probably due to the presence of nutrients within
the matrix (Gu and Chang 2001). Lee et al. immobilized
sensor bacteria to construct a cell array biosensor. They
prepared 20 recombinant sensor bacteria possessing differ-
ent promoters fused with the lux genes, and the cell-agar
mixture was deposited into wells of a cell chip or a
multiwell plate. The sample’s toxicity was analyzed by
bioluminescence measured with a highly sensitive cooled
CCD camera (Lee et al. 2005). Tani et al. used sensor
bacteria possessing an inducible promoter fused with the
luc gene and immobilized in an agarose gel matrix to form
a three-dimensional microfluid network (Tani et al. 2004).
A sample solution was then introduced to induce reporter
gene expression. Bioluminescence at levels detectable by a
1256
CCD camera was produced after the addition of luciferin/
ATP mixtures.
Limitations of whole-cell biosensors
Although many types of bacterial biosensors have been
constructed, as described above, only a few have been
commercialized so far. There are several limitations to be
overcome for the practical use of biosensors. Updated
analytical methods such as gas chromatography mass
spectrometry and liquid chromatography mass spectrometry
can detect analytes in the nanomolar to nanogram-per-liter
range. However, almost no reported bacterial biosensors
can detect analytes at concentrations below 0.1 μM (Van
der Meer et al. 2004). Extensive efforts are necessary to
increase the sensitivity of bacterial biosensors for wide-
spread use.
The diffusion of a substrate and product through the cell
wall or membrane results in a slower response than that of
enzyme-based sensors. The diffusional limitations could be
improved by physical, chemical, and enzymatic approaches.
The lower specificity of a whole-cell biosensor compared
with an enzyme is another limitation to achieving a practical
level of sensitivity. Furthermore, undesirable side-reactions
caused by endogenous enzymes in cells must be minimized.
The major limitation of a whole-cell biosensor made with
recombinant DNA technology involves the time required for
the reporter gene to be transcribed and translated. This delay
may make it difficult to use such sensors for real-time
monitoring.
Future prospects
Many bacterial biosensors using recombinant DNA tech-
nology have been reported and are considered promising
applications in the fields of biotechnology and environ-
mental sciences. The development of portable biosensor
kits will be necessary for biosensors to be used on-site to
monitor environmental pollution. For example, arsenic
contamination of groundwater is a serious problem in
countries such as India and Bangladesh that rely on
millions of tubewells for their drinking water supply. A
simple and sensitive sensor kit is needed to prevent
arsenite-derived diseases in these countries. The practical
application of cell-array biosensors is likely to facilitate the
comprehensive analysis of environmental pollution at
specific sites. High-throughput and sensitive measurements
of specific chemicals, DNA damage, and various stresses
will become possible through the use of innovative micro-
array technology in the near future. Finally, extremely
detailed analyses of samples will be possible with the use of
Appl Microbiol Biotechnol (2007) 73:1251–1258
single-cell biosensors (Tecon and van der Meer 2006). I
anticipate that existing technologies, such as digital
imaging, epifluorescence microscopy, and flow cytometry,
will be sufficient to detect signals from these lone
biosensors. However, it might be difficult to obtain
meaningful information from a single-cell biosensor be-
cause even clonal bacterial populations are rather physio-
logically heterogeneous, which would create difficulties in
obtaining a uniform baseline signal from different cells.
Sophisticated statistical analysis will be needed for the
practical use of single-cell biosensors.
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