Induction of Light Emission by Luminescent Bacteria Treated with UV Light and Chemical Mutagens

J. Appl. Genet. 43(3), 2002, pp. 377-389

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens

Agata CZY¯1, Konrad PLATA2, Grzegorz WÊGRZYN2,3

1 Laboratory of Molecular Biology (affiliated with the University of Gdañsk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Gdañsk, Poland 2 Department of Molecular Biology, University of Gdañsk, Gdañsk, Poland 3 Institute of Oceanology, Polish Academy of Sciences, Gdynia, Poland

Abstract. Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine × 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[a]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.

Key words: bioluminescence, chemical mutagens, Photobacterium leiognathi, Photobacterium phosphoreum, UV irradiation, Vibrio fischeri, Vibrio harveyi.

Received: March 5, 2002. Accepted: May 20, 2002. Correspondence: G. WÊGRZYN, Department of Molecular Biology, University of Gdañsk, ul. K³adki 24, 80-822 Gdañsk, Poland, e-mail: [email protected] 378 A. Czy¿ et al.

Introduction

Light-emitting bacteria are the most abundant and widespread of luminescent or- ganisms (MEIGHEN 1994). Most species of such bacteria live in marine environments (NEALSON 1978). It is well established that luminescent bacteria emit light effectively only when they are at high cell density. This regulation is known as quorum sensing (for a review see SWIFT et al. 1998). However, it has been reported that apart from this regulation, the expression of genes involved in bacterial bioluminescence may be controlled by other factors. For example, production of GroEL and GroES heat shock proteins, which is dependent on the activity of the s32 factor (GROSSMAN et al. 1984), is involved in stimulation of Vibrio fischeri luminescence (ULITZUR,KUHN 1988, ADAR et al. 1992, DOLAN, GREENBERG 1992). When studied in E. coli cells, repression of lux operons from luminescent marine bacteria, V. fischeri and V. harveyi, by the product of the lexA gene has been reported (ULITZUR 1989, SHADEL et al. 1990, CZY¯ et al. 2000a). LexA is a negative regulator of the SOS regulon, and RecA protein activation and cleavage of the LexA repressor upon DNA damage result in SOS response induction (LITTLE,MOUNT 1982). Despite suggestions presented previously (ULITZUR,WEISER 1981, WEISER et al. 1981), it has not been determined whether lux genes of other luminescent bacteria belong to the SOS regulon, and whether quorum sensing and repression by the LexA protein operate independently or these two regulatory systems control expression of lux genes cooperatively. Therefore, in this work we aimed to an- swer these questions by determining the efficiency of light emission by several strains of luminescent marine bacteria after UV irradiation of diluted cultures. Significant UV-mediated induction of luminescence in diluted cultures of V. harveyi was observed previously (CZY¯ et al. 2000a). However, those obser- vations were made after dilution of dense cultures and further long cultivation of bacteria until their luminescence was minimal. Since shortly after dilution of a dense culture there is still a significant amount of luciferase and its substrate in cells and a weak production of autoinducers may occur, light emission decreases gradually. As cell density increases, production of autoinducers also increases. Therefore, after reaching a minimum, the efficiency of light emission per cell increases again (for reviews see MEIGHEN 1994, SWIFT et al. 1998). When effects of UV irradiation are measured at the minimal intensity of luminescence, the influ- ence of the quorum sensing regulation is negligible (CZY¯ et al. 2000a). Repression of the lux operons by LexA (ULITZUR 1989, SHADEL et al. 1990, CZY¯ et al. 2000a) indicates that bacterial luminescence can be enhanced under stress conditions that cause DNA damage. Some previous reports suggested that this may be the case indeed (ULITZUR,WEISER 1981, WEISER et al. 1981). There- fore, we investigated the intensity of bacterial luminescence in the presence of different chemical mutagens. Induction of luminescence by mutagens 379

Bacterial luminescence systems are used in various mutagenicity and toxicity assays (ULITZUR et al. 1980, BULICH,ISENBERG 1981, BEN-ITZHAK et al. 1985, LEVI et al. 1986, ULITZUR,BARAK 1988, SUN,STAHR 1993, BAR,ULITZUR 1994, THOMULKA,LANGE 1995, 1996, LANGE,THOMULKA 1997, PTITSYN et al. 1997, VAN DER LEILE et al. 1997, BEN-ISRAEL et al. 1998, MIN et al. 1999, VERSCHAEVE et al. 1999). The use of these assays is discussed here in the light of regulation of expression of bacterial lux genes.

Material and methods

Bacterial strains and growth conditions The following bacterial strains were used: Photobacterium leiognathi 721, P. leiognathi s-i, P. phosphoreum 8265, P. phosphoreum NZ-1 (all identified by Dr. Anatol Eberhard and kindly provided by Drs. Michael Thomas and Tom Baldwin), Vibrio fischeri ATCC7744 (from the American Type Culture Collec- tion) and V. harveyi BB7 (BELAS et al. 1982). V. harveyi strain was cultured in the BOSS medium (KLEIN et al. 1998), and other strains we grown in the NaCl complete medium, described previously by HASTINGS et al. (1987) and NEALSON (1978). All cultures were kept at 30oC.

Chemical mutagens The following chemical mutagens were used: 2-aminofluorene (2-AF), 4-nitro-o-phenylenediamine (NPD), 2-methoxy-6-chloro-9-(3-(2-chloroethyl) aminopropylamino)acridine × 2HCl (ICR-191), 4-nitroquinolone-N-oxide (NQNO), benzo[a]pyrene (B[a]p) and sodium azide (SA). The doses of used mutagens are shown in Table 1.

Bacterial luminescence assays To estimate effects of UV irradiation on bioluminescence, bacterial strains were grown to a density of about 107 cells per ml. Cultures were diluted 104 times in a fresh medium (total volume of each culture was 10 ml), and incubation was continued for 30 min. Bacteria were harvested by centrifugation (2000 × g, 5 min) and resuspended in an equal volume of 3% NaCl. Following UV irradiation (0-16 J/m2) samples of 0.5 ml each were withdrawn 5 minutes after irradiation and luminescence was measured using a luminometer (Junior, EG&G Berthold). Ef- fects of chemical mutagens on bacterial luminescence were investigated as described above but mutagenic compounds were added (to various, indicated concentrations) directly to diluted cultures of bacteria. Luminescence of bacterial cells was measured using a luminometer (Sirius, Berthold Detection Systems) ev- ery 10 minutes until 240 min after addition of chemicals or in cultures untreated with chemicals. 380 A. Czy¿ et al.

Results

Effect of UV irradiation on bacterial luminescence

To test whether stimulation of luminescence by UV irradiation operates independently of quorum sensing or these two regulatory systems cooperate, we tested effects of UV irradiation relatively shortly after dilution of cultures. A typical curve of luminescence intensity per cell after dilution of a dense V. harveyi culture is shown in Figure 1. We measured effects of UV light on bacteria 30 min after culture dilution, i.e. at the stage when luminescence is less intensive than in the case of dense culture, but still significant. Since maximal UV-mediated induction of lu-

Figure 1. Luminescence of the V. harveyi BB7 culture after its 104-fold dilution (at time 0) in a fresh BOSS medium. Relative luminescence was calculated as counts per one cell. The value measured at time 0 was considered to be 100.

minescence is detected 5 min after irradiation (CZY¯ et al. 2000a, and results not shown), we measured efficiency of light emission at that time. We found effective induction of luminescence under these conditions (Figure 2). When chloramphenicol, an antibiotic that inhibits protein synthesis in bacteria, was added (up to 200 mg/ml) to a bacterial culture 15 min before UV irradiation, no significant increase in luminescence was observed (data not shown), indicating that expression of genes, most probably the lux genes, is necessary to enhance luminescence by UV light. These results, together with our previous report (CZY¯ et al. 2000a), suggest that the UV-mediated induction of light emission by V. harveyi may operate independently of quorum sensing. Such a conclusion was supported by observation of an increase in luminescence intensity in cultures without their Figure 2. Induction of luminescence in bacterial cultures upon UV irradiation. Exponentially growing cultures of P. leiognathi 721 (open circles), P. leiognathi s-i (closed circles), P. phosphoreum 8265 (open squares), P. phosphoreum NZ-1 (closed squares), V. fischeri (open triangles) and V. harveyi (closed triangles) were diluted 104 times in fresh media and incubation was continued for 30 min. Then, bacteria were irradiated with indicated doses of UV. Luminescence was measured 5 min after irradiation. The value measured in a control (non-irradiated) culture of each strain was considered to be 1.

Figure 3. Induction of luminescence of P. leiognathi s-i (panel A) and P. phosphoreum 8265 (panel B) upon treatment of diluted cultures with 2-methoxy-6-chloro-9-(3-(2-chloroethyl) aminopropylamino)acridine × 2HCl (ICR-191). Luminescence of untreated cultures (open circles) and cultures treated with ICR-191 at final concentrations of 0.1 mg/ml (closed circles) or 1 mg/ml (closed squares) is shown. The mutagen was added at time 0, and exponentially growing cultures were diluted 104 times 30 min earlier. Table 1. Induction of luminescence of marine bacteria by chemical mutagens Mutagena Induction of luminescenceb (m/ml) P.l. 721 P.l. s-i P.p. 8265 P.p. NZ-1 V.f. 7744 V.h. BB7 2-AF 0.1 <2 <2 <2 <2 <2 <2 1 <2 3.2 <2 <2 <2 <2 10 6.8 64.6 <2 4.2 <2 INH 100 INH INH 2.2 INH <2 INH NPD 0.0002 <2 <2 <2 <2 <2 <2 0.002 2.5 <2 <2 <2 <2 <2 0.02 3.8 8.4 <2 <2 9.15 <2 0.2 3.8 11.3 <2 <2 2.0 <2 2 3.7 11.5 <2 2.7 INH INH 20 4.2 11.3 2.3 2.4 INH INH 200 <2 <2 231.0 INH INH INH ICR-191 0.0001 <2 <2 <2 <2 <2 <2 0.001 <2 2.7 <2 <2 <2 <2 0.01 <2 4.9 <2 <2 <2 <2 0.1 2.2 5.0 <2 <2 <2 <2 1 12.0 94.8 10.2 2.1 <2 <2 NQNO 0.0001 <2 <2 <2 <2 <2 <2 0.001 2.9 <2 <2 <2 <2 <2 0.01 4.2 <2 <2 <2 <2 <2 0.1 2.2 <2 <2 <2 <2 <2 1 3.5 <2 <2 2.0 <2 <2 10 INH 2.0 INH INH INH IMH 100 INH 2.8 INH INH INH INH B[a]p 0.001 <2 <2 <2 <2 <2 <2 0.01 <2 <2 <2 <2 3.2 <2 0.1 <2 122.3 <2 <2 9.9 <2 1 2.8 15.3 <2 <2 15.3 <2 10 48.1 4.4 3.1 2.0 44.7 <2

NaN3 0.001 <2 <2 <2 <2 <2 <2 0.01 <2 3.5 <2 <2 <2 <2 0.1 <2 4.16 <2 <2 <2 <2 1<2INH 2.3 <2 <2 <2 10 6.39 INH 9.0 3.6 <2 <2 Induction of luminescence by mutagens 383 incubation between dilution and UV-irradiation (data not shown). However, since dilution of a bacterial culture may cause a kind of transient cellular stress, we re- gard the measurements performed with samples from cultures grown for some time after dilution as more conclusive. We tested whether stimulation of luminescence by UV irradiation occurs also in other luminescent marine bacteria (Figure 2). Six strains belonging to four bacterial species were investigated. In all cases we observed a significant induction of luminescence upon treatment of bacteria with UV light, though levels of this induction were different. Again, addition of chloramphenicol before UV-irradiation abolished the induction of luminescence in all tested strains (data not shown). These results strongly suggest that gene-expression-dependent stimulation of light emission by UV irradiation is a general phenomenon rather than an unusual reaction of one or two bacterial species.

Effect of chemical mutagens on bacterial luminescence

We measured the efficiency of luminescence upon treatment of six strains of marine bacteria with different chemical mutagens. It is generally accepted that in assays for testing mutagenic compounds, at least a two-fold increase in a measured parameter (usually the number of mutants) above the value observed in a control experiment should be considered significant. Therefore, in our experiments induction of luminescence was considered significant when equal to or higher than two-fold relative to control cultures. Examples of responses of luminescent bacteria to such a treatment are shown in Figure 3. The results are summarized in Ta- ble 1. Almost all strains responded to the treatment with chemical mutagens by stimulation of luminescence at relatively low concentrations or by its inhibition when higher amounts of these agents were added. The weakest effects were observed in V. harveyi and the greatest stimulation of luminescence occurred in strains of P. leiognathi (Table 1).

a Following mutagens were added to indicated final concentrations into diluted cultures of bacteria: 2-aminofluorene (2-AF), 4-nitro-o-phenylenediamine (NPD), 2-methoxy-6-chloro-9-(3-(2-chloroethyl) aminopropylamino)acridine x 2HCl (ICR-191), 4-nitroquinolone-N-oxide (NQNO), benzo[a]pyrene (B[a]p) and sodium azide (SA). Benzo[a]pyrene and 2-aminofluorene were added together with 4% S9 mix (rat liver microsomal enzymes and cofactors), as described previously (MARON,AMES 1983). b Luminescence of bacterial strains (abbreviations: P.l., Photobacterium leiognathi; P.p., Photobacterium phosphoreum; V.f., Vibrio fischeri; V.h., Vibrio harveyi) in cultures untreated and treated with mutagens was measured 240 min after addition of chemicals. The results represent the ratio of luminescence of treated cultures to that of untreated cultures. INH = inhibition of luminescence upon treatment with a mutagen, which resulted from toxicity of chemicals at particular mutagen concentrations. 384 A. Czy¿ et al.

Discussion

The results presented in this report indicate that UV-mediated stimulation of bacterial luminescence is a general phenomenon, not restricted to one species. A significant increase in luminescence was found in all strains of marine luminescent bacteria tested (Figure 2). Since for two species, V. fischeri and V. harveyi, LexA-mediated repression of lux operons has been shown (ULITZUR 1989, CZY¯ et al. 2000a), it is likely that a similar regulation operates also in P. leiognathi and P. phosphoreum. UV-mediated stimulation of luminescence was observed both relatively shortly after dilution of a dense bacterial culture (Figure 2) and at the stage of minimal luminescence of the diluted culture (CZY¯ et al. 2000a). These results suggest that two processes of stimulation of lux operon expression (quorum sensing and inactivation of the LexA repressor) operate independently. This hypothesis is supported by our observation of an increase in luminescence intensity in cultures without their incubation between dilution and UV-irradiation. The double regulation of expression of bacterial lux genes may arise from the presence of two independent promoters, one being regulated by quorum sensing and the other belonging to the SOS regulon. In fact, the presence of two promoters in the lux operon of V. fischeri has already been demonstrated (ULITZUR et al. 1997, ULITZUR 1998a). Of these two promoters, only one is regulated by the quorum sensing mechanism (ULITZUR 1998b). One might argue that the UV-mediated induction of luminescence could be due to formation of free radicals by UV or by reduction of H2O2, generated by UV, which was not reversed by catalase. In fact, it has been demonstrated that UV-irradiation my lead to generation of active oxygen species, and that luminescence resulting from the activity of bacterial luciferase may be enhanced in the presence of H2O2 (HASTINGS,BALNY 1975, WATANABE,NAKAMURA 1976, WATANABE,HASTINGS 1987, ICHIKI et al. 1994, ZHANG et al. 1997). However, although our results do not negate involvement of active oxygen forms in enhancement of luminescence, the hypothesis that UV-mediated stimulation of light emission in diluted bacterial cultures arises solely from physical effects of irradiation is unlikely. We demonstrated that this stimulation is abolished in the presence of a translation inhibitor, chloramphenicol, thus this process in strongly dependent on effective gene expression. Moreover, using different concentrations of catalase, added to bacterial cultures, we were not able to observe inhibition of UV-stimulated luminescence (our unpublished results). Apart from UV irradiation, luminescence of marine bacteria tested in this study was stimulated by chemical mutagens (Table 1 and Figure 3). Induction of bacterial luminescence after treatment of cultures with DNA-damaging or DNA synthesis-inhibiting agents was demonstrated previously (ULITZUR,WEISER 1981, WEISER et al. 1981). Interestingly, under these conditions, luminescence of dark mutants (with dysfunction of regulatory element(s) rather than with muta- tions in lux genes) was also induced. It has been hypothesized that mutagenic Induction of luminescence by mutagens 385 agents through their interactions with DNA may cause configuration changes of the double helix resulting in derepression of transcription of the lux operon (ULITZUR,WEISER 1981). However, in our opinion, results presented in this report, together with findings described previously by others (ULITZUR 1989, SHADEL et al. 1990), support the proposal that bacterial lux operons belong to SOS regulons. The proposal of regulation of lux genes’ expression by agents inducing the SOS response may have implications for the use of bacterial luminescence systems in mutagenicity and toxicity tests. Although many mutagenicity assays based on non-luminescent bacteria were developed previously, including the most widely used Ames test (MARON,AMES 1983, MORTELMANS,ZEIGER 2000), most of them are excellent for testing whether a given chemical is a mutagen, but their sensitivities are too low for testing environmental samples, in which concentrations of mutagens are very low. The use of bioluminescent bacteria in detection of toxic chemicals was pro- posed over 20 years ago (ULITZUR et al. 1980, BULICH,ISENBERG 1981), and various assays have been described since then (see for example: THOMULKA,LANGE 1995, 1996, LANGE,THOMULKA 1997). In those studies, the toxicity of different chemicals was determined by a relatively simple method based on measuring a de- crease in bioluminescence after addition of toxic compounds. However, toxicity assays should be distinguished from mutagenicity tests. Toxic agents are not al- ways mutagens, and mutagenic chemicals are often toxic only at relatively high concentrations (compare Table 1). Therefore, although these bioluminescence assays are simple, they can be useful in detection of toxic substances rather than mutagens that occur in natural habitats at concentrations too low to provoke seri- ous toxic effects in bacterial cells. In several tests, fusions consisting of lux operons under control of one of LexA-repressed promoters are used (BAR,ULITZUR 1994, PTITSYN et al. 1997, VAN DER LELIE et al. 1997, BEN-ISRAEL et al. 1998, MIN et al. 1999, VERSCHAEVE et al. 1999). Strains bearing such fusions emit light upon contact with SOS response-inducing agents, which may be a sensitive and quick indica- tion of the presence of mutagenic compounds in the tested sample. However, these fusions are constructed in Escherichia coli or Salmonella enterica serovar Typhimurium. This is a disadvantage in direct testing of samples from certain habitats, for example marine waters. These bacteria live normally in completely different habitats and addition of marine water samples to their cultures might induce a stress response per se. Moreover, survival of E. coli and S. enterica in marine water is impaired relative to marine bacteria. Therefore, for monitoring of marine habitats an organism that naturally lives in these habitats should be used (CZY¯ et al. 2000b). A very useful group of genotoxicity tests employing light emission by bacteria is that based on detection of restoration of luminescence of dark mutants, including the commercially available Mutatox test (ULITZUR et al. 1980, BEN-ITZHAK 386 A. Czy¿ et al. et al. 1985, LEVI et al. 1986, ULITZUR,BARAK 1988, SUN,STAHR 1993). These assays are often significantly (up to 103 times) more sensitive than the Ames test, and have been demonstrated to be useful in environmental studies (BRENNER et al. 1993a, b, 1994, BELKIN et al. 1994). Their only disadvantage seems to be that effective restoration of luminescence of dark mutants caused by mutagens is usually observed about 24 h after addition of tested compounds. In this report we show that mutagen-mediated induction of luminescence of diluted cultures of wild-type bacterial strains is effective 6 h after addition of a mutagenic compound. Therefore, one might consider the use of wild-type luminescent bacteria, espe- cially P. leiognathi strains, for modification and/or improvement of mutagenicity assays. In the light of the proposal that bacerial lux operons belong to SOS regulons, this suggestion may be of particular interest.

Conclusions

Results presented in this report, together with previous reports, indicate that bacterial luminescence systems, apart from being regulated by quorum sensing, are stimulated by factors inducing the SOS response. It is likely that lux operons of most, if not all, luminescent bacteria are repressed by the lexA gene product, and thus belong to SOS regulons. The two processes of stimulation of lux operon expression mentioned above, operate independently rather than coordinately. Such a double regulation of expression of genes involved in bacterial luminescence has implications for the use of luminescent bacteria in mutagenicity assays.

Acknowledgments. We are grateful to Michael THOMAS and Tom BALDWIN for providing bacterial strains, and to Shimon ULITZUR for discussions. This work was supported by the University of Gdañsk (project grant no. BW-1190-5-0033-1 to A.C.), Polish State Committee for Scientific Research (project grant no. 6 P04G 033 19 to Hanna SZPILEWSKA, Institute of Oceanology of the Polish Academy of Sci- ences), Provincial Nature Protection Fund in Gdañsk (project grant no. WFOŒ/D/210/3/2001 to G.W.), Institute of Oceanology of the Polish Academy of Sciences (task grant to G.W.), and Foundation for Polish Science (subsidy 14/2000 to G.W. and a stipend to A.C.).

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