Mutagenesis, Vol. 14, No. 4, 351-356,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Evaluation of transcriptional fusions with green fluorescent protein versus luciferase as reporters in bacterial mutagenicity tests
School of Biological Sciences, The Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia
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Abstract |
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A bacterial plasmid was constructed on which the regulatory region of the umuC gene of Escherichia coli was fused to the coding sequence of the green fluorescent protein gene (gfp) from the jellyfish Aequorea victoria. Escherichia coli AB1157 strains carrying the plasmid emitted fluorescence in the presence of mutagens that induce the SOS DNA repair system. Data on tests with nitrosoguanidine, methylmethane sulphonate and UV radiation (254 nm) are presented. Although fluorescent detection using this system was not as rapid or sensitive as a similar luminescent equivalent (umuC–luxAB), the gfp reporter system was more robust. Escherichia coli umu gene induction was also analysed in Salmonella typhimurium TA1537 cells following plasmid transfer and exposure to the same range of mutagens. There was no significant difference in sensitivity between the two species. These preliminary results will provide the basis for development of mutagenicity test systems useful in the testing of complex mixtures, such as environmental samples, and the investigation of physiological parameters influencing spontaneous mutagenesis in bacteria.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A number of different types of reporter genes have recently been investigated for their ability to facilitate detection of gene expression under different experimental conditions. One such reporter, the lacZ gene, requires a colourimetric enzyme assay to detect ß-galactosidase (Huisman and D'Ari, 1981
; Shinagawa et al., 1983). This type of system has been used to detect environmental mutagens (Oda et al., 1985; Reifferscheid et al., 1991), genotoxic chemicals (Quillardet et al., 1985; Ong et al., 1987) and disinfectants and their metabolites (Sakagami et al., 1988). Another widely used reporter system is based on the lux genes from marine bacteria which mediate a luminescent reaction. lux genes have been used in the detection of soil ecotoxicity (Paton et al., 1995a), chemical and toxicological characterisation of river water (Guzzella and Galassi, 1993), the detection of bioavailability of heavy metals (Selifonova et al., 1993; Paton et al., 1995b) and to monitor bacteria in soil (Meikle et al., 1992). Both the lux and ß-galactosidase systems require cofactors/substrates for reactions in which assayable products act as the reporters for detection of gene activity.
The cloning of a third novel reporter gene of interest is that of green fluorescent protein (gfp) from the jellyfish Aequorea victoria. Green fluorescent protein has led to new developments in reporter gene studies because its detection does not require any cofactors or substrates for fluorescence to occur. Cells can thus be monitored in situ without disruption. Although eukaryotic in origin, mutant forms of gfp have been shown to be successfully expressed in prokaryotic cells at high levels (Cormack et al., 1996), thus making it a species-independent reporter gene. One major advantage gfp provides is its relative stability in living cells for several days or even weeks. All of these factors make gfp a more robust reporter gene than many others in current use (see Cariello et al., 1998).
Recently we have reported on the construction of a strain in which the luxAB luciferase genes have been fused to the Escherichia coli umuDC DNA repair operon (Justus and Thomas, 1998). This construct was capable of detecting various concentrations of both chemical and physical mutagens via the emission of light in a dose-dependent manner. It also provided a much simpler and rapid test for mutagenicity than other currently available tests, but with comparable sensitivity. The following paper describes the construction of a umuC–gfp gene fusion and its subsequent use in detecting agents that promote mutagenic DNA repair in E.coli and Salmonella typhimurium. It also presents data comparing the merits of lux (Justus and Thomas, 1998) versus gfp for the detection of mutagenic agents, with a view to providing suggestions to interested researchers.
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Materials and methods |
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Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are listed in Table 1
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Media and growth conditions
Bacteria were grown in nutrient broth (Oxoid CM1) or on nutrient agar (Oxoid CM3), both supplemented with the addition of 50 µg/ml ampicillin (Sigma A951) shaken at 200 r.p.m. at 37°C.
DNA cloning
Methods for plasmid DNA isolation, restriction endonuclease digests, DNA ligation, alkaline phosphatase treatment and agarose gel electrophoresis techniques followed standard protocols (Sambrook et al., 1989). Cells were transformed as described by Sambrook et al. (1989) with the following modifications: cells were heat shocked at 37°C for 10 min and then grown in nutrient broth. DNA was isolated from agarose using agarase from Pseudomonas atlantica according to the manufacturer's instructions (Boehringer Mannheim).
PCR
PCR amplification of the gfp gene sequence was conducted as described by Matthysse et al. (1996).
Epifluorescence microscopy
Photomicrographs were taken of cells as described by Matthysse et al. (1996).
umuC–gfp gene fusion construction
The 700 bp gfp PCR fragment was ligated to a 7 kb HindIII–EcoRI fragment from plasmid pSE117 (Figure 1). Escherichia coli GW2100 cells were transformed with this ligation mixture and transformants selected on nutrient agar with ampicillin.
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gfp–umuC gene fusion confirmation
Plasmid preparations of colonies that resulted from ligation were PCR amplified using gfp primers to confirm the presence of the gfp gene (data not shown). Colonies were also screened for fluorescence using epifluorescence microscopy.
Luminescent and fluorescent detection methods
Luminescent assays were performed as described by Justus and Thomas, (1998). Stationary phase cells were obtained for experiments by allowing cells to grow up overnight. Logarithmic phase cells were obtained by diluting overnight culture cells 1:10 and regrowing them for 2 h at 37°C prior to use in experiments. During fluorescent assays bacterial cell cultures in either logarithmic phase or stationary phase (2 ml) were treated with mutagen and then added to 18 ml of nutrient broth or 0.85% saline with ampicillin. For UV-irradiated cells this involved irradiating cells in Falcon 60x15 mm tissue culture dishes with regular stirring. All other mutagens were added to this 2 ml of cells. This mixture was then dispensed at 150 µl/well into 96 Uni-well microtitre plates (Elkay) and incubated at 37°C with shaking. Fluorescence was measured using a Fluoroskan Ascent by Labsystems using the Ascent computer software package v.2.2, with an emission filter of 510 nm and excitation filter of 485 nm. Measurements were taken at three time intervals, time 1 being fluorescence prior to incubation, time 2 at 4.5 h and time 3 at 24 h. All fluorescent values are presented as the means (error bars show standard deviations) of 96 cultures grown in parallel to obtain a statistically more accurate representation of mutation rate.
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Results |
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umu gene induction in stationary phase cells and logarithmic phase cells
To observe if there is a difference in transcriptional induction of the umu operon during mutagenesis of logarithmic phase cells compared with stationary phase cells, cultures in both growth phases were tested following mutagen treatment (Figures 2–4
). The fluorescent signal from stationary phase cells did not appear to differ significantly from those in logarithmic phase (Figures 2–4). The chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) induced a dose-dependent increase in fluorescence at 1.5, 3.5 and 7 µg/ml (Figure 2). There was no significant increase in fluorescence at 0.1 µg/ml when compared with the 0 dose control (Figure 2). When another chemical mutagen, methylmethane sulphonate (MMS), was assayed cells showed no significant difference in fluorescence from the 0 dose control and 13 µg/ml MMS but responded in a dose-dependent manner at 325 and 650 µg/ml (Figure 3). At a higher dose of 1300 µg/ml a lower level of fluorescence was observed, most likely due to cellular toxicity (Figure 3). The physical mutagen UV irradiation failed to induce a significant difference in fluorescence from the 0 dose control at 1 J/M2 but at 3 and 6 J/M2 a dose-dependent response was seen (Figure 4). The results described here suggest that when cells are exposed to a mutagen in nutrient broth umu transcriptional induction occurs whether cells are in logarithmic or stationary phase.
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, 0 µg/ml MNNG; 
, 0.1 µg/ml MNNG; x, 1.5 µg/ml MNNG; 
, 3.5 µg/ml MNNG; 
, 7 µg/ml MNNG.
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Reporter gene comparisons
Comparative sensitivity of the two reporter systems was established by exposing strains carrying either the umuC'::gfp or umuC'::luxAB fusion plasmids to low doses of mutagens. The results show that lower threshold mutagen doses were required to induce umuC'::luxAB transcription than those required to induce umuC'::gfp transcription (Figures 2–4
and Tables 2–4). This and a previous study (Justus and Thomas, 1998) showed that the lux system responded positively to 13 µg/ml MMS, 0.1 µg/ml MNNG and 1 J/M2 UV, whereas gfp transcription was not induced at these doses. Thus the lux reporter gene appears to be a more sensitive reporter than gfp. However, when assays were conducted over longer periods of time (i.e. 24 h) the signal from the gfp reporter system increased with time whilst there was a marked decrease in lux-derived luminescence (Figures 2–5). This demonstrates the more robust nature of the gfp system compared with lux, which is very dependent on cell physiology at the immediate time of assay due to its shorter half-life. gfp may thus be more suitable for assays where the mutagen dose being assayed is comparatively high and a rapid result is not crucial.
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, TA1537/pTJ10, 0 J/m2 UV; 
, TA1537/pTJ10, 1 J/m2 UV.The effect of starvation on reporter gene expression
It is known that the lux gene is dependent upon cells being metabolically active for successful gene expression to occur (Justus and Thomas, 1998
). To discover if the same is true of gfp gene expression cells were assayed in 0.85% saline. The results shown in Figures 6 and 7 suggest that cells do not require cultivation in a nutrient-rich medium for a dose-dependent response to a mutagen to occur. However, there is the requirement that cells be in logarithmic phase (Figure 6) and not stationary phase (Figure 7) for reporter gene induction to occur in saline.
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Comparisons of E.coli umu gene expression in E.coli and S.typhimurium
Luminescent assays suggest that the E.coli umu operon is expressed at a slightly greater magnitude in S.typhimurium cells than in E.coli cells (Tables 2–4
), as shown by the amount of luminescence seen in cells prior to challenge with a mutagen. However, despite this apparent `advantage', the S.typhimurium strain used in this study does not consistently appear to be more sensitive to lower mutagen doses than the E.coli strain, as measured by the ratio of fluorescence emitted from treated versus untreated cells. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The aim of this study was to investigate the usefulness of green fluorescent protein activity as a reporter in bacterial tests for mutagenicity. Previous work from this laboratory (Justus and Thomas, 1998
) has shown that the luxA and luxB genes from Vibrio harveyi can be successfully fused to bacterial DNA repair genes to provide a sensitive and semi-quantitative assay for the presence of DNA-damaging agents. One drawback with this system, however, is the need for cells to be in a metabolically active state, together with the transient state of the luminescent signal that begins to decline after 24 h. This means that for the test to be useful samples would need to be processed in the laboratory under quite invariant conditions. The current study sought to determine whether an alternative reporter activity (green fluorescent protein) could provide a more stable signal without compromising the sensitivity of the test system and at the same time decrease the necessity for relying on metabolically active systems or substrate addition. The longer term aim of the latter strategy would be the creation of a system more amenable to use in environmental testing of complex mixtures. Results in which bacterial strains carrying either umuC':: luxAB or umuC'::gfp plasmids were compared directly for their responses to the presence of the DNA-damaging agents MNNG, MMS and UV showed that the luxAB construct luminesced at lower doses than those required to cause fluorescence in the gfp construct. This appears to indicate that luminescence is a more sensitive reporter system in this instance than fluorescence and would therefore be more useful in the potential detection of weak mutagenic agents. The distinct advantage of the gfp system though does appear to be the robust and long lasting nature of the signal. When cells carrying either reporter construct were left in the presence of mutagens for 24 h before reading, fluorescent signals could still be detected easily, whereas luminescence was not. This raises the prospect of being able to use gfp-based testing under conditions where sensitivity, for whatever reason, is not an issue but extended exposure to mutagenic agents is required
The gfp-based reporter system showed no detectable variation in response when using cells from different growth phases in nutrient broth, unlike the lux system. Dose-dependent increases in fluorescence were observed for each of the mutagens tested, however, when cells were starved induced fluorescence was only seen in logarithmic phase cells. This could partly be due to the use of sequestered nutrients, but a similar response was not seen in the lux-based system (Justus and Thomas, 1998). This is one advantage of the gfp reporter system compared with lux (Meikle et al., 1992; Justus and Thomas, 1998). As a result, GFP may be a more suitable reporter for detection of high levels of mutagens in environmental samples, as often nutrient levels in such samples may be low.
Salmonella typhimurium is known to have much weaker native Umu protein activity than E.coli (see Thomas and Sedgwick, 1989) although the two operons are regulated similarly, hence, the study sought to compare any host cell effects on the sensitivity of the light-based reporter systems. Although basal and induced level expression is slightly higher in S.typhimurium than in E.coli, sensitivity was virtually identical. This indicates that the reporter systems might be used with equal success in either species, opening up the prospect of creating niche-specific reporter strains more suitable for testing different types of environmental samples.
In conclusion, this study has shown that a second light-based reporter system based on green fluorescent protein can be used to successfully detect physical and chemical mutagens in bacteria. gfp provides a more robust signal than lux-based systems, which can be read easily in a standard fluorimeter with appropriate filters, thus lowering the time required to generate results. While it does seem to lack some sensitivity compared with the lux system, the minimal processing required for the test, together with its use in a microtitre plate format, raises the possibility of running multiple parallel culture tests which would provide an abundance of data for statistical analysis. Future work will be aimed at ascertaining how well this approach will work for detecting low mutagen doses and for investigating parameters that perturb the spontaneous mutation rate within a population.
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Acknowledgments |
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The authors are grateful to Serina Stretton for gfp PCR primers and to both Amanda Goodmans's and Nick McClure's research groups for assistance with gfp. Research in S.T.'s laboratory was supported by the Australian Research Council and Flinders University. T.J. is the recipient of an Australian Post-graduate Research Award.
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Notes |
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1 To whom correspondence should be addressed. Tel: +61 8 8201 3144: Fax: +61 8 8201 3015; Email: sue.thomas{at}flinders.edu.au
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References |
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Received on July 21, 1998; accepted on March 18, 1999.
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