RESEARCH LETTER
The potential applications of SOS-lux biosensors for rapid
screening of mutagenic chemicals
Hani A. Alhadrami1,2 & Graeme I. Paton1,3
1
Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, UK; 2Faculty of Applied Medical Sciences, King Abdulaziz
University, Jeddah, Saudi Arabia; and 3Remedios Limited, Balgownie Technology Centre, Aberdeen, UK
Correspondence: Hani A. Alhadrami,
Faculty of Applied Medical Sciences, King
Abdulaziz University, P.O. Box 80324, Jeddah
21589, Saudi Arabia. Tel.: +966 5055
45275; fax: +966 2 6400000 Ext. 20171;
e-mail: [email protected]
Received 26 February 2013; accepted 10
April 2013. Final version published online 1
May 2013.
DOI: 10.1111/1574-6968.12156
Editor: Geoffrey Gadd
MICROBIOLOGY LETTERS
Keywords
SOS-lux biosensors; Salmonella assay;
mutagenicity; bioluminescent bacteria;
polycyclic aromatic hydrocarbons.
Abstract
The environmental fate and potency of mutagenic compounds is of growing
concern. This has necessitated the development and application of rapid assays
to screen large numbers of samples for their genotoxic and carcinogenic effects.
Despite the development of biosensors for genotoxicity assessment, these have
not been calibrated against traditional microbial bioassays. In this study, assays
using the SOS-lux-marked microbial biosensors Escherichia coli K12C600 and
E. coli DPD1718 were refined and optimised to screen selected mutagenic
chemicals. The response of the biosensors was compared with the mutagenic
response of the traditional Salmonella mutagenicity assay. For the chemicals
tested (acridine, B[a]A, B[a]P, chrysene, mitomycin C and sodium azide),
E. coli DPD1718 was consistently more sensitive than E. coli K12C600. The
biosensors were of comparable sensitivity to the Salmonella assay but were
more rapid, reproducible and easier to measure. These data validate the adoption of optimised assays making use of microbial biosensors for routine screening of test chemicals.
Introduction
Chemical analysis alone is a poor strategy for assessing the
mutagenicity of compounds found in the environment.
Such compounds, once activated, increase the rate of
mutation in cells. Chemical analysis may identify the presence and quantity of such compounds but does not reflect
the potency to biological receptors. To effectively measure
mutagenicity, there is a requirement for assays with a clear
end points and capability of screening large numbers of
samples (pure compounds, mixtures and environmental
samples).
Luminescence-based bacterial biosensors can detect the
mutagenic mode of action of certain chemicals (Gu &
Chang, 2001) and are simple to apply, sensitive and easy
to measure (Van Der Meer & Belkin, 2010). The
SOS-lux-based microbial biosensors have a promoterless
lux-operon (luxCDABE) under control of the SOSdependent col promoter (Rettberg et al., 1999). When a
mutagenic mode of action is activated, this leads to an
FEMS Microbiol Lett 344 (2013) 69–76
increase in the concentration of luciferase and, as a consequence, bioluminescence.
The Salmonella assay (Ames assay) is the most widely
used bacterial assay for screening potential mutagenic compounds (Pereira et al., 2010). There is a correlation
between mutagenicity as measured by the Ames assay and
carcinogenicity in mammals (Josephy et al., 1997). The
assay uses a number of Salmonella strains with specific
mutations that disable the cells from synthesising histidine.
New mutations at the site of these pre-existing mutations
can restore the gene’s function and allow the cells to resynthesise histidine. Enumeration of a cultured lawn of these
mutated colonies in the absence of histidine enables a
quantitative assessment of mutagenicity (Mortelmans &
Zeiger, 2000). The assay is simple to perform but requires
high replication, a wide range of controls, extensive culturing and time-consuming enumeration.
Polycyclic aromatic hydrocarbons (PAHs) are classified
by the European Union and U.S. Environmental Protection
Agency as priority environmental pollutants due to their
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
70
mutagenic impact on humans (Song et al., 2009). Certain
PAHs are biologically inactive and require liver metabolism
before activation to a mutagenic form (Shimada & Fujii-Kuriyama, 2004). Most bacteria are unable to metabolise
such chemicals via cytochrome P450; therefore, the assay
requires the exogenous addition of the mammalian metabolite (Maron & Ames, 1983). This metabolite is typically
delivered to the assay in the presence of NADP, glucose-6-phosphate and phosphate buffer (S9 mix).
Polycyclic aromatic hydrocarbons can cause several
types of mutations such as frameshift mutations,
base-pair-substitution mutation and transition mutation
(Perez et al., 2003). Frameshift mutations are a consequence of the addition or deletion of one or two
consecutive base pairs in the DNA sequence of a gene,
resulting in misreading mRNA and synthesis of a nonfunctional protein (Sadava et al., 2006). Base-pair-substitution
mutation is a result of the abnormal replacement of a single-base nucleotide with another nucleotide of the genetic
materials (Mortelmans & Zeiger, 2000). Transition mutation is when a replacement of purine base (i.e. adenine and
guanine) with another purine or a replacement of a pyrimidine base (i.e. thymine, cytosine and uracil) with another
pyrimidine occurs (Sadava et al., 2006). The Salmonella
isolates can be modified to suit specific receptors.
In this study, the performance of the SOS-lux-based
microbial biosensors for the screening of mutagenic
chemicals was compared with the traditional Salmonella
assay (the Ames assay), and a critical comparative evaluation of assays sensitivity and performance was made.
H.A. Alhadrami & G.I. Paton
Salmonella typhimurium TA98, TA100 and TA102 were
obtained from Molecular Toxicology Inc. (Boone, NC) and
maintained according to standard protocols (Mortelmans
& Zeiger, 2000). Each sample was tested in triplicate in the
absence and the presence of the mammalian liver homogenate (S9 mix derived from Aroclor 1254-exposed rats and
obtained from Molecular Toxicology Inc.). In brief,
Salmonella strains TA98, TA100 and TA102 were grown
overnight in 150-mL Erlenmeyer flask containing 25 mL
Oxoid nutrient broth at 37 °C in an orbital shaker at
150 r.p.m. with appropriate antibiotics (25 lg mL 1 ampicillin for TA98 and TA100, and 2 lg mL 1 tetracycline for
TA102). The cultures were incubated until they reached
an absorbance of 1.0 at 660 nm (corresponding to
1–2 9 109 CFU mL 1). Two millilitre of melted top agar
supplemented with histidine and biotin solution was distributed into sterile glass tubes and placed in a 45 °C water
bath. For the mutagenicity assay conducted without S9
mix, 100 lL of the test chemical and 100 lL of the tester
strain were added, gently mixed by vortexing and poured
onto the surface of minimal glucose agar plate. The plates
were gently tilted and rotated to obtain an even distribution, placed onto a level surface to solidify and incubated at
37 °C for 48 h. Following the incubation, the revertant colonies were enumerated on a Gallenkamp colony counter.
To conduct the assay with the S9 mix, 500 lL of the previously prepared S9 mix (Maron & Ames, 1983) was
added to the top agar along with the test chemical and the
tester strain. Appropriate reagent and negative controls
(Maron & Ames, 1983) were included to enumerate the
spontaneous revertants.
Materials and methods
Test solutions
Mutagenicity testing was performed against a range of
doses of acridine, benzo (a) anthracene (B[a]A), benzo
(a) pyrene (B[a]P), chrysene, mitomycin C (MMC) and
sodium azide. All chemicals were purchased from Sigma
(St. Louis, MO). Standards (except MMC) were dissolved
in 100% v/v spectrophotometric grade DMSO. MMC was
dissolved in MilliQ water. Dilutions were performed to
obtain appropriate assay concentrations, and these were
tested against appropriate controls (in equivalent to
DMSO concentration and MilliQ water). The selection of
the chemicals doses was based upon data published by
McCann et al. (1975) and Madill et al. (1999).
Mutagenicity assessment using the Salmonella
mutagenicity assay
The standard plate incorporation procedure described by
Maron & Ames (1983) was used for the Ames assay.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
Mutagenicity assessment using the SOS-lux
microbial biosensors
An aliquot (25 mL) of biosensor strains Escherichia coli
K12C600 and E. coli DPD1718 was grown overnight on LB
media at 37 °C on an orbital shaker at 150 r.p.m. in the
presence of the appropriate antibiotics (50 lg mL 1 ampicillin for E. coli K12C600 and 30 lg mL 1 chloramphenicol
for E. coli DPD1718) (Rettberg et al., 1999; Vankemmelbeke
et al., 2005). Overnight cultures were diluted 1:50 in LB
broth and grown at 37 °C until they reached their preoptimised CFU (1–2 9 109 CFU mL 1). A negative control of
100 lL MilliQ water, a DMSO reagent control or 100 lL
tested chemical were mixed with 900 lL of overnight culture in 3mL luminometer cuvettes. Bioluminescence was
measured using a Jade bench-top luminometer (Labtech
International, Uckfield, UK), and each sample was tested in
triplicate. To determine whether mutagenicity occurred,
the test sample was incubated for a 300min period, and the
maximum induction (relative to controls) that occurred
between 180 and 300 min was recorded.
FEMS Microbiol Lett 344 (2013) 69–76
Applications of SOS-lux biosensors
Statistical analysis
Statistical analysis was performed using MINITAB 15 for
Windows. Normality testing and equal variances were
carried out to assess normality. Analysis of variance
(ANOVA) and t-test were applied to assess the differences
between the bioluminescence readings of the biosensors.
Correlation tests (Pearson product-moment or Spearman
rank-order) assessed the correlation between the Ames
assay and the biosensors. A result with P 0.05 was
considered significant. The ‘twofold increase rule’ was
applied to the Salmonella assay data to define whether a
test compound was mutagenic (Maron & Ames, 1983).
For the biosensors, a compound was considered a mutagen if there was a significant difference between the bioluminescence readings of the negative control and the test
compound during the incubation period. If the bioluminescence values decreased during the incubation time, the
compound was considered more likely to be a cytotoxic
(Rettberg et al., 1999).
The lowest observed adverse effective concentration
(LOAEC) was the dose at which there was a significant
(P 0.05) measure of mutagenicity relative to the control values for both of the assays.
Results and discussion
Mutagenicity response using the Salmonella
mutagenicity assay
A significant mutagenic response of Salmonella strain
TA98 was recorded in response to B[a]A tested at
500 lg mL 1 in the absence of S9 mix (Table 1). In
contrast to this, B[a]P and chrysene only had a significant mutagenic impact on the Salmonella strains tested
in the presence of S9 mix (Table 1). When TA100 was
exposed to acridine at concentrations of 3 lg mL 1,
5 lg mL 1 and 10 lg mL 1, a significant mutagenic
response was observed (Table 1). A significant mutagenicity was reported for sodium azide tested with TA98
and TA100 (at 3 lg mL 1, 5 lg mL 1 and 10 lg mL 1)
and TA102 (at 10 lg mL 1) (Table 1). This remark substantiated that sodium azide is as direct-acting potent
mutagen when tested with the Salmonella assay, which
correlates with the observation of Nilan et al. (1973) who
reported that sodium azide was a base-pair-substitution
mutagen.
For the Salmonella strains to be responsive to B[a]A, B
[a]P and chrysene, the exogenous addition of S9 mix was
required (Table 1). Significant dose-dependent mutagenic
effects occurred for B[a]P when exposed to Salmonella
strains TA98 and TA100 (Table 1). This confirmed that
B[a]P caused a base-pair-substitution mutation as it
FEMS Microbiol Lett 344 (2013) 69–76
71
reverted TA100, and a frameshift mutation as it reverted
TA98. These observations were in agreement with data
published by Unger & Guttenplan (1980). The exogenous
addition of S9 is used because B[a]P requires activation
by cytochrome P450 (CYPs) enzymes to form bay-region
epoxides through two activation steps: The first step
would involve the formation of B[a]P-7,8-oxide by the
action of CYP. The second step would be the formation
of B[a]P-7,8-diol by microsomal epoxide hydrolase
(Gelboin, 1980). The recombinant human CYP1B1 is
acknowledged to be more active than CYP1A1 in catalysing oxidation of B[a]P to B[a]P-7,8-diol with human
liver epoxide hydrolase being present in the reaction mixture. Data previously published (Shimada & Fujii-Kuriyama, 2004) confirmed that CYP1B1 was essential for the
first-step oxidation of B[a]P at the 7,8-position; hence, it
played a more critical role than CYP1A1 in the tumorigenesis caused by B[a]P.
Salmonella strains TA98 and TA102 were responsive to
B[a]A in the presence of the S9 mix (Table 1). It has
been acknowledged that B[a]A causes frameshift mutations and transition mutation (McCann et al., 1975).
Chrysene had a significant mutagenic effect on TA98
at all tested doses except 5 lg mL 1 (Table 1). Chrysene
at concentration of 50 lg mL 1 was mutagenic to
Salmonella strain TA100 (Table 1). Chrysene is a causative agent of frameshift mutations, and this was
approved by the reversion of TA98 only in the presence
of the S9 mix. Similar results were reported by Malachova (1999).
Acridine, chrysene, B[a]A and B[a]P caused a mutagenic response to the Ames assay at concentrations
between 20 and 200 lg mL 1 (Madill et al., 1999). Previous data (McCann et al., 1975; Perez et al., 2003)
reported the mutagenic effects of these compounds in the
presence of S9 mix.
There was a degree of inconsistency in the dose
response of the Ames assay, but using the collated data,
the LOAEC was predicted (Table 2). Fundamentally,
there may not be a clear dose response such as that
observed for chronic and acute exposure assays, because
once the critical mutagenic dose is reached, the effect
may remain constant. At elevated doses, a cytotoxic effect
may occur, and this could inhibit or indeed cause confusion in the detection of mutagenicity. In addition to
problems in interpreting the data, the actual assay could
cause artefacts because the concentration of hydrophobic
compounds present in the agar may not correlate with
the causal dose because the agar could disrupt the
bioavailability of the test sample. For future assays, a
measure of the diffusion of the test chemical may be a
better indicator of the exposure dose rather than the
amendment concentration added.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
72
H.A. Alhadrami & G.I. Paton
Table 1. The number of reverse mutants of Salmonella typhimurium TA98, TA100 and TA102 by B[a]A, B[a]P, chrysene, sodium azide and
acridine at various doses tested with and without S9 mix
Number of revertants/plate (mean SE)*
Compound
B[a]A
Positive control¶
B[a]P
Positive control¶
Chrysene
Positive control¶
Sodium azide
Positive control¶
Acridine
Positive control¶
Dose
(lg mL 1)†
0
2.5
5
10
25
50
0
1
3
5
7
10
0
2.5
5
10
25
50
0
0.5
1.5
3
5
10
0
0.5
1
3
5
10
Without S9 mix
TA98
13
20
21
20
17
26
1339
23
17
14
18
16
15
1088
14
12
12
12
9
16
1428
13
16
19
35
27
35
6472
27
25
13
19
14
22
1336
With S9 mix
TA100
1
2
2
2
1
3§
0§
0‡
1
0
1
2
0
27§
2‡
3
3
2
1
3
0§
2‡
4
1
3§
2§
1§
0§
4‡
3
6
3
7
5
0§
‡
81
78
58
93
86
96
160
72
54
71
68
71
71
725
73
74
84
110
67
79
287
125
211
306
416
437
499
488
90
192
197
309
289
274
557
TA102
1
8
12
2
5
1
0§
1‡
0
3
4
5
5
9§
15‡
11
3
9
1
9
0§
6‡
7
7§
7§
14§
2§
0§
40‡
11
20
10§
8§
16§
0§
‡
388 362 381 322 402 300 1572 NT**
NT**
NT**
NT**
NT**
NT**
NT**
326 348 348 347 334 323 1240 117 167 207 168 203 360 611 245 67 61 84 80 99 845 TA98
9
32
12
12
3
3
0§
‡
8‡
10
7
11
8
39
0§
4‡
8
13
4
6
10§
0§
20‡
5
11
3
6
12
0§
21 52 54 57 42 42 2008 15 225 477 345 346 326 1948 21 56 36 47 47 55 2008 NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
TA100
1
3§
2§
5§
7§
3§
0§
0‡
8§
12§
18§
2§
2§
1§
1‡
10§
3
2§
6§
3§
0§
‡
167 329 260 325 298 215 904 57 431 956 1283 1432 1730 1005 180 318 354 353 350 366 2239 NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
TA102
10
26
9
16
4
20
0
0‡
28§
7§
33§
79§
35§
14§
3‡
15
15
5
6
15§
0§
‡
349 730 605 736 721 718 906 NT**
NT**
NT**
NT**
NT**
NT**
NT**
458 540 547 535 587 503 914 NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
NT**
9‡
19§
19
34§
8§
7§
0§
23‡
10
15
16
22
10
0§
*Number of histidine revertants per plate: mean values of at least three plates standard error (SE).
Concentration based on 100 9 15-mm Petri dish containing 20–25 mL of MG agar.
‡
Numbers in italic, boldface and underlined represent the number of spontaneous revertants colonies for each strain. The spontaneous revertant
ranges (revertants/plate without S9 mix) were as follows: TA98 (13-77), TA100 (67-173) and TA102 (117-530).
§
Numbers in boldface represent twofold increase or more in the number of revertant colonies over the solvent controls (spontaneous revertants),
which was an indication of a significant mutagenic response.
¶
Positive control: TA98 – daunomycin (60 lg mL 1); TA100 – sodium azide (15 lg mL 1); TA102 – mitomycin C (5 lg mL 1).
**NT: compound was not tested against the strain(s), B[a]P was recommended to be tested only with TA98 and TA100 (McCann et al., 1975).
Acridine and sodium azide were recommended to be assayed in the absence of the S9 mix (Madill et al., 1999).
†
Mutagenicity response using the SOS-lux
biosensors E. coli K12C600 and E. coli DPD1718
Mitomycin C caused a significant induction of the
luminescence of the SOS-lux-marked biosensor E. coli
K12C600 and was considered a mutagen. This was
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
adopted as a positive control (Fig. 1). The responses of
the biosensor to acridine tested at 1 and 3 lg mL 1 and
to 5 lg mL 1 chrysene were significantly different
(P = 0.01 and 0.02, respectively) to the reagent control
(MilliQ water) defining these as having mutagenic modes
of action (Fig. 1). There was a significant mutagenic
FEMS Microbiol Lett 344 (2013) 69–76
73
Applications of SOS-lux biosensors
Table 2. Lowest observed adverse effective concentration values
(lg mL 1) for each of the chemicals tested for individual assays
(n = 3)
Compound
Chrysene
Acridine
B[a]A
B[a]P
Sodium azide
MMC
Biosensors strains
Salmonella mutagenicity
strains
E. coli
K12C600
E. coli
DPD1718
TA98
a
b
5.32
0.31e
1.92 h
0.09
0.06f
0.07i
1.84
0.02
j
m
0.02
m
TA100
c
0.86
TA102
d
28.12
NR
*1.24 g
1.72 h
2.12 h
1.97 h
i
i
0.02
0.05
*2.73j
*0.92k
*6.08 l
Adopted as positive control
The values represent the concentrations at which there was a significant difference from the reagent control (P 0.05). For the Ames
assay, each of the assays was conducted using S9 mix except for this
preceded with *. Values with the same letter following the value are
not significantly different from one another (P 0.05). NR designates no mutagenic response at the doses tested.
response (P = 0.02) to B[a]A tested at 50 lg mL 1 and
to sodium azide (P = 0.01) tested at 0.5, 1, 1.5, 3 and
5 lg mL 1 (Fig. 1). There was no response to B[a]P in
the presence or the absence of S9 mix (data not shown).
Mitomycin C, acridine and chrysene caused significant
induction of E. coli DPD1718 (Fig. 2) defining these chemicals as mutagenic. B[a]P tested at 1 lg mL 1 in the presence
of S9 mix also caused a significant increase in luminescence.
These findings confirmed that E. coli DPD1718 responded
to the DNA cross-link mode of action of MMC, acridine
and chrysene (Fig. 2).
The biosensor responses reflected those of the Ames
assay but were less responsive to B[a]P. This failure to
respond to B[a]P and other such chemicals (that are
acknowledged to be mutagenic) could be on account of
several reasons central to the operational activities of
these biosensors and the assay procedures adopted. B[a]P
and similar molecules may be incapable of inducing the
SOS-DNA repair system of E. coli K12C600. The SOS
responses are stimulated by disrupting and arresting the
DNA, which in turn inhibits the cell division and leads to
the accumulation of single-stranded DNA (ssDNA) (Janion, 2008). Chemicals that are direct inducers of the SOS
system such as MMC and methyl methane sulphonate
(MMS) will be more mutagenic to the biosensor.
The biosensor E. coli K12C600, unlike the Salmonella
mutagenicity strains, does not carry rfa (increase of cell
wall permeability) and uvrB (the excision repair system
for DNA) mutations. Therefore, the cell wall of E. coli
K12C600 is less permeable than the cells in the Ames
assay resulting in reduced transportation of hydrophobic
molecules. Indeed, this was the least sensitive of the
assays tested. As with the Ames assay, the bioavailability
FEMS Microbiol Lett 344 (2013) 69–76
of hydrophobic molecules within the assay procedure
could be a key constraint on its performance.
Rettberg et al. (2001) used the pPLS1 plasmid in E. coli
K12C600 and constructed another SOS-lux biosensor by
introducing pPLS1 plasmid into S. typhimurium TA1535
(which carries rfa mutation). The resultant S. typhimurium TA1535 had greater sensitivity for the detection of
hydrophobic compounds when compared with E. coli
K12C600 (Rettberg et al., 2001).
These biosensors in which mutagenic compounds damage DNA and activate the reporter genes fused with the
SOS repairing genes may be prone to the occurrence of
false-negative responses when inhibition of metabolism is
a consequence of cytotoxicity (Sørensen et al., 2006). To
address this, the user must consider adopting a wide
selection of chemical doses to ‘range-find’ the responses
and differentiate cytotoxicity from mutagenicity. In this
study, LOAEC values were calculated to drive a more
relative comparative value of assay sensitivity (Table 2).
Comparative evaluation of the SOS-lux
biosensors and the Ames assay
The SOS-lux biosensors were adequate for the detection
of the mutagenicity of DNA-damaging substances at the
range of concentrations associated with the Ames assay.
An example is given for B[a]P: in the Ames assay, the
LOAEC was between 0.02 and 0.05 lg mL 1 for
S. typhimurium TA98 and TA100 (Table 2). Similarly, the
LOAEC for E. coli DPD1718 was 0.07 lg mL 1 (Fig. 2
and Table 2). There was no significant difference between
any of these assays. With acridine, the LOAEC for the
biosensors E. coli DPD1718 and E. coli K12C600 was 0.06
and 0.31 lg mL 1, respectively, both significantly lower
than the value for the Ames assay (1.24 lg mL 1 for
S. typhimurium TA100) (Table 2). The biosensor E. coli
DPD1718 was never less sensitive to the test chemicals
than the Ames assay (Table 2).
For the collated data, correlation analysis confirmed a
relatively close correlation between the induction of
the SOS system as measured in the biosensor E. coli
K12C600 and mutagenesis as measured in the Ames assay
(P 0.05). Nevertheless, no significant correlation was
reported between E. coli DPD1718 and the Ames assay
(P = 0.98). Nor did a physicochemical factor such as
Kow, aqueous solubility or Henry’s Law Constant aid in
explaining the relationship between the mutagenic values
for each of the assays (Debnath et al., 1992). It has, however, been shown by several authors (i.e. Quillardet &
Hofnung, 1993; Mersch-Sundermann et al., 1994) that
the results of SOS chromotest are comparable to those of
the Ames assay with regard to the genotoxic mutagenic
potency and that 60–70% of genotoxic substances
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
74
H.A. Alhadrami & G.I. Paton
Acridine
Chrysene
25
4.0
24
Bioluminescence (RLU)
Bioluminescence (RLU)
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
23
22
21
20
2.2
2.0
0
2
4
6
8
19
10
0
10
Concentration (μg mL–1)
20
B[a]A
50
MMC
26
Bioluminescence (RLU)
Bioluminescence (RLU)
40
3000
28
24
22
20
18
16
14
12
10
30
Concentration (μg mL–1)
2500
2000
1500
1000
500
0
0
10
20
30
40
50
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
Concentration (μg mL–1)
Concentration (μg mL–1)
Sodium azide
22
Bioluminescence (RLU)
21
20
19
18
17
16
15
14
0
2
4
6
8
10
Concentration (μg mL–1)
Fig. 1. Mutagenicity response of the SOS-lux microbial biosensor Escherichia coli K12C600 to acridine, chrysene, B[a]A, MMC and sodium azide.
Error bars represent standard errors of the mean of triplicate measurements.
identified by the SOS bioassays are also carcinogenic to
mammals.
The SOS-lux-marked biosensors may have several key
benefits when compared with the Ames assay including
the following: (1) an assessment of mutagenicity can be
made in 3 h, while the Ames results take at least 48 h;
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
(2) the SOS-lux biosensors can detect a range of
mutagenic chemicals with different DNA-damaging mechanisms using the same biosensor strain; (3) the simultaneous measurement of cell number/CFU and light
emission of the biosensors allow discrimination between
mutagenicity and cytotoxicity of the test substance;
FEMS Microbiol Lett 344 (2013) 69–76
75
Applications of SOS-lux biosensors
Chrysene
90
70
80
Bioluminescence (RLU)
Bioluminescence (RLU)
Acridine
80
60
50
40
30
20
60
50
40
30
20
10
0
70
0
20
40
60
Concentration (μg
80
10
100
0
mL-1)
20
40
60
80
100
Concentration (μg mL-1)
B[a]P With S9 mix
MMC
100
3500
Bioluminescence (RLU)
Bioluminescence (RLU)
3000
80
60
40
20
0
2500
2000
1500
1000
500
0
0
20
40
60
80
100
Concentration (μg mL-1)
0.0
0.1
0.2
0.3
0.4
0.5
Concentration (μg mL-1)
Fig. 2. Mutagenicity response of the SOS-lux microbial biosensor Escherichia coli DPD1718 to acridine, chrysene, B[a]P tested in the presence of
S9 mix and MMC. Error bars represent standard errors of the mean of triplicate measurements.
(4) temporal monitoring of the biosensor allows a kinetic
interpretation of the response, which can be related to
the form and mode of action of the chemical; and (5) the
SOS-lux biosensors are capable of rapid screening of large
sample numbers in a microplate format.
While there is a need for a sensitive, rapid and costeffective bioassay for monitoring mutagenicity, it is clearly
shown that no single bioassay can be applied in isolation
to provide a definite and comprehensive assessment of
the mutagenic hazard in samples under investigation. The
SOS-lux biosensors cannot replace the role of direct measurement of carcinogenic effects in animals or detection
of chromosomal aberrations in humans. However, such
an approach can be employed as a cost-effective screening
tool prior to a more established technique.
Conclusion
The Ames Salmonella assay has allowed a sensitive and
accurate screening of molecules for their potential
mutagenicity. Although it is laborious, it can be streamFEMS Microbiol Lett 344 (2013) 69–76
lined through the use of automated colony counting to
enumerate histidine revertants colonies. However, Salmonella strains are classified as human pathogens, and in
some countries, there are significant restrictions in their
use and applications. The two SOS-lux biosensors had
several practical advantages over the traditional assays
including procedural simplicity, a rapid and unambiguous
result, ease of measurement, rapid tabulation of exposure
concentration and in vivo analysis without cell disruption.
If the cell wall permeability of the biosensors can be
enhanced, through assay refinement, then such an
approach could transform rapid screening of potentially
mutagenic samples.
Acknowledgement
The authors would like to thank Dr. Petra Rettberg and
Dr. Ying Zhang for providing the biosensors strains.
Dr. Matt Aitkenhead is also acknowledged for developing
the colony counter software. The government of Saudi
Arabia is acknowledged for funding this project.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
76
References
Debnath AK, Debnath G, Shusterman AJ & Hansch C (1992)
A QSAR investigation of the role of hydrophobicity in
regulating mutagenicity in the Ames test: 1. Mutagenicity of
aromatic and heteroaromatic amines in Salmonella
typhimurium TA98 and TA100. Environ Mol Mutagen 19:
37–52.
Gelboin HV (1980) Benzo[alpha]pyrene metabolism, activation
and carcinogenesis: role and regulation of mixed-function
oxidases and related enzymes. Physiol Rev 60: 1107–1166.
Gu MB & Chang ST (2001) Soil biosensor for the detection of
PAH toxicity using an immobilized recombinant bacterium
and a biosurfactant. Biosens Bioelectron 16: 667–674.
Janion C (2008) Inducible SOS response system of DNA repair
and mutagenesis in Escherichia coli. Int J Biol Sci 4: 338–344.
Josephy PD, Gruz P & Nohmi T (1997) Recent advances in
the construction of bacterial genotoxicity assays. Mutat Res
386: 1–23.
Madill REA, Brownlee BG, Josephy PD & Bunce NJ (1999)
Comparison of the Ames Salmonella assay and Mutatox
genotoxicity assay for assessing the mutagenicity of
polycyclic aromatic compounds in porewater from
Athabasca oil sands mature fine tailings. Environ Sci Technol
33: 2510–2516.
Malachova K (1999) Using short-term mutagenicity tests for
the evaluation of genotoxicity of contaminated soils. Soil
Sediment Contam 8: 667–680.
Maron DM & Ames BN (1983) Revised methods for the
Salmonella mutagenicity test. Mutat Res 113: 173–215.
McCann J, Choi E, Yamasaki E & Ames BN (1975) Detection
of carcinogens as mutagens in the Salmonella/microsome
test: assay of 300 chemicals. P Natl Acad Sci USA 72:
5135–5139.
Mersch-Sundermann V, Schneider U, Klopman G &
Rosenkranz HS (1994) SOS induction in Escherichia coli and
Salmonella mutagenicity: a comparison using 330
compounds. Mutagenesis 9: 205–224.
Mortelmans K & Zeiger E (2000) The Ames Salmonella/
microsome mutagenicity assay. Mutat Res, Fundam Mol
Mech Mutagen 455: 29–60.
Nilan RA, Sideris EG, Kleinhofs A, Sander C & Konzak CF
(1973) Azide – a potent mutagen. Mutat Res 17: 142–144.
Pereira TS, Gotor GN, Beltrami LS, Nolla CG, Rocha JAV,
Broto FP, Comellas LR & Vargas VMF (2010) Salmonella
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
H.A. Alhadrami & G.I. Paton
mutagenicity assessment of airborne particulate matter
collected from urban areas of Rio Grande do Sul State,
Brazil, differing in anthropogenic influences and polycyclic
aromatic hydrocarbon levels. Mutat Res, Genet Toxicol
Environ Mutagen 702: 78–85.
Perez S, Reifferscheid G, Eichhorn P & Barcel
o D (2003)
Assessment of the mutagenic potency of sewage sludges
contaminated with polycyclic aromatic hydrocarbons by an
Ames fluctuation assay. Environ Toxicol Chem 22:
2576–2584.
Quillardet P & Hofnung M (1993) The SOS chromotest:
a review. Mutat Res 297: 235–279.
Rettberg P, Baumstark-Khan C, Bandel K, Ptitsyn LR &
Horneck G (1999) Microscale application of the
SOS-LUX-TEST as biosensor for genotoxic agents. Anal
Chim Acta 387: 289–296.
Rettberg P, Bandel K, Baumstark-Khan C & Horneck G (2001)
Increased sensitivity of the SOS-LUX-Test for the detection
of hydrophobic genotoxic substances with Salmonella
typhimurium TA1535 as host strain. Anal Chim Acta 426:
167–173.
Sadava D, Heller HC, Orians GH, Purves WK & Hillis DM
(2006) Life: The Science of Biology, 8th edn. The Courier
Companies, Inc., Philadelphia.
Shimada T & Fujii-Kuriyama Y (2004) Metabolic activation of
polycyclic aromatic hydrocarbons to carcinogens by
cytochromes P450 1A1 and 1B1. Cancer Sci 95: 1–6.
Song Y, Li G, Thornton SF, Thompson IP, Banwart SA, Lerner
DN & Huang WE (2009) Optimization of bacterial whole
cell bioreporters for toxicity assay of environmental samples.
Environ Sci Technol 43: 7931–7938.
Sørensen SJ, Burmølle M & Hansen LH (2006) Making
bio-sense of toxicity: new developments in whole-cell
biosensors. Curr Opin Biotechnol 17: 11–16.
Unger J & Guttenplan JB (1980) Kinetics of benzo[a]pyrene
induced mutagenesis in a highly sensitive Salmonella/
microsome assay. Mutat Res 77: 221–228.
Van Der Meer JR & Belkin S (2010) Where microbiology
meets microengineering: design and applications of reporter
bacteria. Nat Rev Microbiol 8: 511–522.
Vankemmelbeke M, Healy B, Moore GR, Kleanthous C,
Penfold CN & James R (2005) Rapid detection of colicin
E9-induced DNA damage using Escherichia coli cells
carrying SOS promoter-lux fusions. J Bacteriol 187:
4900–4907.
FEMS Microbiol Lett 344 (2013) 69–76