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Anal. Chem. 1992, 64, 1755-1759
Bioluminescence Detection System of Mutagen Using Firefly Luciferase Genes Introduced in Escherichia coli Lysogenic Strain SooMi Lee,?Masayasu Suzuki,? Michiyo KumagaiJ Hideo Ikeda,t Eiichi Tamiya,? and Isao Karube’J Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1Komaba, Meguro-ku, Tokyo, 153 Japan, and Institute of Medical Science, University of Tokyo, P.O. Tanakawa, Tokyo, 108 Japan
A rapld and convenient mlcroblai sensing sydem for mutagena was developed bawd upon the induction of prophage from Escherkh/a coll lysogenic straln and bioiuminscence. The sydem con8Med of lysogenic E. cd/encoding firefly luclferawgenes and a photodetectbnsydem. Measurement of mutagen mitomycin C was achleved by measuring the luminescence Intensity emitted from E. co// iywgenlc drain for the recombinant phage in the presence of luminescence substrates. Approximately 1 h afler addltlon of mitomycln C, the iumhscence began to be observed, and 3 h afler, it attained a ievel of 2 times greater than that of 1h. Irradlatlon with uitraviolet light air0 produced ilght based on Induction of phago from the E. cd/ lysogenic drain for the recombinant phage. On the other hand, when nonmutagenlc toxic compound8 like sodium azide were added to the reactlon medlum, luminescencewa8 not observed. MitomycinC could be detected wnhln 1 h with thi8 sensing system, at concentrations down to lo2 ng/assay.
INTRODUCT10N Many kinds of chemicalsexist in the environment; of special note, carcinogenshave become a social problem. With respect to environmental care and human health concerns, development of rapid and simple monitoring methods for carcinogens are needed. Although carcinogenicity tests using animals like monkeys and mice are the most trusted methods, in practice long-term carcinogenicitytesta with whole animals are time-consuming and demand resources. The mutagenic activity of carcinogens has recently been confirmed in a great number of chemicals,’ and corrections between carcinogenicityand mutagenicity provide useful tests for potential carcinogens. A number of mutagenic tests using microorganismsfor preliminary screening of carcinogenshave been developed. The “Ames test” and “rec-as~ay”,~J using Salmonella typhimurium histidine auxotroph,recombination deficient strain, and Bacillus subtilis (Rec-), respectively, have been applied for prescreening tests of chemical carcinogens. These methods are more rapid and simple than the carcinogenicity test using animals. However, these methods still need a lengthy incubation with bacteria and complicated procedures. Author to whom correspondence should be addressed. Center for Advanced Science and Technology. t Institute of Medical Science. (1)Ames, B. N.; Durston, W. E.; Yamasaki, E.; Lee, F. D. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2281-2285. (2)Ames,B. N.;Lee, F.D.Durston, W. E.Proc.Natl. Acad. Sci. U.S.A. 1973, 70, 782-786. (3)Kada, T.; Tutikawa, K.; Sadaie, Y. Mutat. Res. 1972,16, 165-174. + Research
0003-2700/92/0364-1755$03.00/0
Our group has already developed various types of microbial sensors for the preliminary screening of mutagens.44 These sensors consist of immobilized microorganismsand an oxygen electrode which determined the respiration activity of microorganisms. However, since the sensors measure changes of the respiration activity a t the sensing part of the electrode, the response properties are dependent not only on the nature of the microorganism but also on the diffusion and other reaction properties of the substrate. Moreover, as the responses of these sensors were based on measurements of respiration activity of the microorganism, it could be affected by even nonmutagenic compounds like bactericides as in the case of a high concentration. Moreau et al. proposed another microbialdetection method, the “Inductest”.7 The test detecta various kinds of mutagens by their ability to induce prophage X from lysogenic E. coli cells. Although the Inductest has not been validated as extensively as the Ames test, the two appear to be valuable complementary assays. The method is also particularly valuable for determining the nature of DNA damage and for establishing a correlation between the ability of a chemical to produce DNA damage in bacteria and ita potency in causing cancer in animals. On the other hand, bioluminescenceassays concerned with firefly luciferase reaction have been developed as highly sensitive and rapid detection methods in various fields.8 In this study, the genesencodingluciferase from firefly were cloned into phage X and furthermore, it was infected into E. coli, which was integrated in chromosomal DNA of E. coli. By combination of the phage induction test and photodetection system, therefore, an extremely rapid and simple microbial sensing system for mutagens is expected. We describe here the determination of mutagens by measuring the luciferase activity released by phage induction utilizing E. coli lysogenic strain for recombinant phage and photomultiplier tube.
EXPERIMENTAL SECTION Preparation of Phage-Containing Firefly Luciferase Genes. The plasmid gene encoding firefly luciferase was kindly donated by Professor Deluca’s group (University of Calif~rnia).~ Phage X gtl0 was used for vector cloning the luciferase genes. At (4)Karube, I.;Matsunaga,T.;Nakahara,T.; Suzuki, S.;Kada,T. Anal. Chem. 1981,53, 1024-1026. (5)Karube, I.; Nakahara, T.; Matsunaga, T.; Suzuki, S. Anal. Chem. 1982, 54, 1725-1721. (6)Karube, I.; Sode, K.; Suzuki, M.; Nakahara, T. Anal. Chem. 1989, 61,2388-2391. (7)Moreau, P.;Bailone, A.; Devoret, R. Proc. Natl. Acad. Sci. U.S.A. 1976, 73,3700-3704. ( 8 ) Beckers, B.; Lang, H. R. M.; Schimke, D.; Lammers, A. Eur. J . Clin. Microbiol. 1985, December, 556-561. (9) Dewet, J. R.; Wood,K. V.; Deluca, M. Mol. Cell. B i d . 1987, Feb, 725-131. 0 1992 American Chemical Society
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luciferase genes
1 gtl0
ligation
Lu434
'VI
EcoRl
infection into E.coli
(7)
DNA of E.colf
I
1
n
t-
(8) Figure 1. Preparation of recombinant lysogenic E. coli containing
firefly luciferase genes.
first, the X gtl0 DNA was digested at the EcoRl site located in the imm434 region. The restricted fragment containing luciferase genes which were cloned using a vector, pRIT2T and X promoter (X,,R) were isolated by electrophoresis using agarose gel, and the luciferase genes with promoter were ligated in the EcoRl site. The recombinant phage was proved by the plaque hybridization method,1°and bioluminescencebased on the activity of luciferase which was expressed in phage-infected E. coli cells. Construction of E. coli Lysogenic Strain for the Recombinant Phage. Since the above recombinant A phage has no ability to produce repressor, the infected X DNA is impossible to lysogenize by single infection. Thus, another phage X imm434 containing wild type repressor gene as helper was infected with the recombinant phage. The lysogenic E. coli containing luciferase genes was prepared by infecting recombinant X phage (1.2 X 109mL-') and X imm434 (1.7 X 109rnL-l) simultaneously into E. coli as shown in Figure 1. The lysogenic strain carrying the recombinant phage was screened by both plaque test and immunity test. The strain thus obtained is lysogenic for both the recombinant phage and A imm434 phage, since both types of phages are produced by spontaneous induction of the lysogenic cell. Properties of Host Cell a n d Cultivation of Microorganism. Host cell for h phage (E.coli GY5026) was provided by Professor R. Devoret (Section de Radiobiologie Cellulaire, Laboratoire d'Enzymologie, C.N.R.S., 91190 Gif-sur-Yvette, France). It was derived from E. coli K12 and was undertaken with enuA and uurB mutation to increase permeability of cell membrane and susceptibility to drugs. In all experiments, an overnight culture in L-broth at 37 "C was diluted 1:lOO into fresh same medium and incubated under aerobic conditions at 37 "C until the logarithmic phase was reached and just before the mutagens were added. L-broth was composed with trypton (10 g L-l), yeast extract (5g L-I), and NaCl(10 g L-l); solid-state broth was supplemented with agar (12 g L-I). Growth, estimated by the increase of absorbance at 660 nm, was measured in a UV/vis spectrophotometer (JASCO uvidec-510). Chemicals. Polytrypton, yeast extract, and agar were purchased from Difco Laboratories. Luciferin synthesized by Sigma Chemical Co. was used. Mitomycin C was from Waco Pure Chem. Ind., and ATP (5'-adenosinetriphosphoricacid) was from Tokyo Kasei Chem. Ind. Other reagents were commercially available or of laboratory-grade materials. Deionized water was used in all procedures. Construction of Sensing System and Measurement of Bioluminescence. The scheme of the sensing system is illustrated in Figure 2. The system consists of a photomultiplier, amplifier, photon counter (HAMAMATSU Photonics, Co., Japan) and personal computer (Nippon Electric Co. PC-9801VM). (10) Benton, W.D.; Davis, R. W. Science 1977, 196,180
Figure 2. Schematic diagram of the sensing system: (1) photomultiplier, (2) amplifier, (3) photon counter, (4) personal computer, (5) sample tube, (6) dark box, (7) InJector,(8) alr pump.
A sample tube containing 200 pL of cell suspensions with 5 mM ATP was fixed in front of a photomultiplier tube. A 100-pL portion of 1 mM of luciferin solution was injected with a microsyringe into the sample tube; the response changes were recorded and analyzed by the computer. The bioluminescence intensity was expressed as number of photons per second. Assay Procedure. Mytomicin C was dissolved in distilled water at varying concentrations of solution and was diluted with 0.1 M phosphate buffer to pH 7.0. Given concentrations of mytomicin C were added in cell suspensions of logarithmic phase and incubated under aerobic conditions at 37 O C in the L-broth. Phage induction by ultraviolet light was conducted by cultivation under aerobic conditions with shaking at 37 O C after irradiation for 8 s using 30-W UV light. Above 8 s the cells could not be grown, and below 8 s phage induction by UV could not be conducted. Principle of Bioluminescence by Phage Induction Using the E. coli Lysogenic Strain for the Recombinant Phage. Generally, since the prophage is repressed by its repressor, it cannot be grown in the lysogenicstrain. But if the strain is treated with UV light or chemical compounds which cause damage to the DNA, an activated RecA protein (protein responsible for DNA repair) is produced, which subsequently decomposes the repressor protein of phage. As a result, the growth of phage is induced in the cell, consequently E. coli is lysed. With the induction of phage, further transcription of luciferase genes occurs, so the enzyme luciferase is produced. Firefly luciferase produces light in the presence of ATP and luciferin as a substrate.I1
luciferin + ATP + Mg2+
-
luciferaseluciferyl-AMP
+ 0,
luciferase
luciferaseluciferyl-AMP
-
+ ppi
luciferase + oxyluciferin + AMP + CO, + hv (560 nm)
Therefore, mutagenicity of a chemicalcompoundcan be estimated by measuring the luciferase activity released by phage induction (see Figure 3).
RESULTS AND DISCUSSION Pattern and Time Course of Luminescence Intensity by UV Irradiation on the Sensing System. Figure 4a-c shows the luminescence pattern following UV irradiation of the sensing system and luciferin injection. The luminescence pattern in the early state of UV irradiation shows a gentle response curve, which means there was a start of cell lysis by ~
~
~
(11) Thierry, A.; Chicheportiche,R. Appl.Microbiol.Biotechnol.1988, 28,199-202.
ANALYTICAL CHEMISTRY. VOL.
64.NO. 17. SEPTEMBER 1. 1992 1787
n4
*s*
O*' DNA of
**\ E.coll
prophage A eooralning luciferase ~ e n e s
recombinant DNA
G9-Sor A phwe
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phage induction. But with the lapse of time, a faster lumineacence reaction was obtained in complete lysis condition. The most attractive aspect of these results is that the stateof a phage induction of lyscgenicstraincan be anticipated in a short time directly from this luminescence pattern. Thetimecouraeoflumineacenceintensityby UVirradiation of the sensing system was also investigated. As shown in Figure 5, the luminescence was detected within 30 min of incubationafter UV irradiation. Themaximum luminescence intensity was observed after 210 min of incubation, and afterward it decreased gradually. Typical Reuponue Cuweof the SensingSystem toward Mitomycin C. Detection of the mutagen mitomycin C was then investigated using the sensing system. Figure 6 show a typical response curve of the sensing system toward m i t omycin C. This response curve was obtained after a I-h incubation with the addition of mitomycin C to the reaction medium (mitomycin C concentration: 10 pglassay). M i t e mycin C is well-known to induce growth of prophage in the lysogenic strain as well as an anticancer agent acting as DNA damaging agent. Within 1h of incubation, the luminescence was obtained following injection of luciferin and ATP, while no luminescence was observed from the mitomycin C untreated cells. After the injection of luciferin solution, the luminescence intensity reached a maximum value immediately, and the response returned to the base line within 10 8.
Time Course of Luminescence Intenclity toward Mitomycin C on the Sensing System. Time course of the luminescence intensity toward mitomycin C on the sensing system was also examined. As shown in Figure 7. the luminescencewas observed within 1 h of incubation, and the maximum luminescence intensity was obtained after 3 h of incubation. The luminescence increase observed by the sensing system reflects expression of luciferase genes by induction of prophage in the E. coli lysogenic for the recombinant phage. The addition of mitomycin C caused the E. coli strain to induct phage A together with the lysis of cell. It was expected that the decreased luminescence after 3 h of incubation is due to the inhibition of luciferase activity by productareleased by lysisofcells. Therefore, theoptimum incubation time for detection of mutagen is about 3 h. It has heen suggested that the addition of mitomycin C as well as UV irradiation causes damage of the lysogenic E. coli DNA
< 2 >
or produce some structural changes. These series of changes in the promotor region appear to initiate the transcription of luciferase genes, either directly or indirectly through altering theabilityofthe DNAto bind with therepressor. Therefore, it was considered that the mutagenicity of chemical compounds can be estimated hy determining luminescence intensity utilizing this sensing system. Effect of Mitomycin C Concentration on the Luminescence Intensity. The effect of mitomycin C concentrations on the luminescence intensity was also examined. Figure 8 shows the relationship between mitomycin C concentrations and the luminescence intensity observed 3 h after addition of mitomycin C. A good correlation wan obtained between mitomycin C concentration and the luminescence intensity. Mitomycin C could be detected using the microbial sensing system below 102 nglassay. For each measurement. average values of three determinations of single concentrations were taken; the relative standard deviation was about 7% a t a concentration of IO2 ng/assay. On the other hand, the response of the sensing system to nonmutagenictoxic compounds wasalso investigated. Chlorampbenicolandsodium azide were usedas toxic compounds. Chloramphenicol is known as an antibiotic that could inhibit protein synthesis, while sodium azide is a bactericide. When these chemicals were incubated in the reaction medium, the luminescencewasnot observed. These results suggested that this sensing system can possibly selectively detect mutagens which could damage DNA. In addition, the effect of various kinds of culture broth on the luminescence intensity was examined. When the E. coli lysogenic strain for the recombinant phage was cultivated in nutrition-rich broth like L broth, the luminescence intensity was 5 times larger than compared to A broth. The effect of mitomycin C on the microorganism and luminescence intensity itself was also examined (see Figure 9, curves 1and 2). In this study, firefly luciferase introduced recombinant E. coli prepared previously in our laboratory was used.'* As shown in this result, no effect of mitomycin C on the microorganism and lumineacence intensity was observed. Also in this study, a more effective detection method of luminescence by treatment of lysozyme was (12) Lee. S.M.;Swuki, M.;Tamiya. E.;Karube. I. A d . Chim. Acto 1991,244,201-206.
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10
0
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(
n 0
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60 (
20
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Time ( sec I Flgurr 6. Typical response curve of the sensing system toward mitomycin C: (1) without mitomycin C; (2) with mitomycin C (10 pg/ assay). Other conditions as in Figurs 4.
100
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-0
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Time ( h ) Flgure 7. Dependence of maximum lumlnescence intenstty on time of Incubation of mitomycin C with the senslng system. Experimental conditions as in Figure 6.
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Time ( sec 1 Flgurr 4. Change of lumlnescence intenslty pattern following luclferin and ATP Injection afler UV lrradlation of the sensing system: (a) 30min Incubation after UV Irradlatlon; (b) 60-min Incubation after UV Irradlatlon; (c) 2 10-mln Incubationafter UV lrradlatlon. Luminescence substrate: iuclferln, 1 mM, 1OOpL. ATP 5 mM, lOOpL. Ceilvoiumes: 200 pL.
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I
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Flgurr 8. Effect of mitomyclnC concentratlons on the luminescence intenstty: (1) 102 ng/assay; (2) lo3 ng/assay; (3) lo4 ng/assay.
60 0 3 e
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I
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Time ( min ) Flgurr 6. Dependence of maximum lumlnescence intenstty on time of Incubation followlng UV irradiationof the sensing system. Experimental conditions as In Flgure 4.
attempted. As shown in Figure 9, curve 3, the luminescence intensity was increased approximately 10 times as compared to lysozyme-untreated whole cells, and the inhibition of luminescence by that was not observed at all. The luminescence increase by addition of lysozyme is due to a more
effective determination of luciferase activity by the lysis of the cells. Therefore, more sensitive and rapid determination of mutagens was anticipated by pretreatment of lysozyme just before determination. Compared to other mutagen detection methods by phage induction, the detection limit of a mutagen like mitomycin C was scarcely different. But this conventional assay method needs a very long detection time and complicated procedure, and it can determine prophage induction by counting infectivecenters formed after overnight incubation. Thus this assay method requires a more rapid and simple procedure. The need for a rapid determination of mutagens may be met by using our sensing system; it takes only less than 1h to measure luciferase activity based upon prophage induction. It is a simple method without complicated procedures. It is also capable of differentiating mutagen from nonmutagenic toxic compounds. In addition, we
ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992
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By applying this improved system to the mutagen detection, it is expected that a wider range of mutagens may be detected more sensitively. In conclusion, this sensing system provides useful data for a rapid prescreening of potentially carcinogenic compounds existing in the environment. This rapid and simple detection method will enable testing of anticancer agents and new drugs or chemical agents that react directly or indirectly with the genetic materials within limited times and allow the comprehensive study of carcinogens.
(c
0
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Time ( sec ) Flgw 9. Effect of mitomycinC on the microorganismand luminescence intensity itself: (1) without mitomycin C; (2) with mitomycin C (100 ng mL-I). Recombinant E. coli encoding firefly luciferase genes was cultivated just before effect of the mitomycinC was measured under aerobic conditions at 37 O C for 6 h in the L-broth. Cell volumes: 200 pL. Luciferin: 1 mM, 100 pL. (3) Effect of lysozyme on the luminescenceintensity. Lysozyme concentration: 200 pg mL-I. Other conditions as in (1) and (2).
have recently constructed an improved sensing system, which contains the luciferase-introduced lysogenic E. coli and possesses a higher capacity of phage induction as a host cell.
ACKNOWLEDGMENT We wish to acknowledge Ms.E. N. Navera for her helpful assistance in editing this paper and Professor H. Ikeda and Miss M. Kumagai for useful discussions and assistance during the experiments.
RECEIVED for review November 13, 1991. Accepted April 14,1992. Registry No. Mitomycin C, 50-07-7; luciferase, 9014-00-0.
