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Improved bacterial SOS promoter∷lux fusions for genotoxicity detection
Mutation Research 466 Ž2000. 97–107 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres
Improved bacterial SOS promoter
DiÕision of EnÕironmental Sciences, the Fredy and Nadine Herrmann Graduate School of Applied Science, The Hebrew UniÕersity of Jerusalem, Jerusalem 91904, Israel b DuPont Central Life Sciences, P.O. Box 80173, Wilmington, DE 19880-0173, USA c Swarthmore College, Swarthmore, PA 19081-1397, USA Received 15 September 1999; received in revised form 8 December 1999; accepted 14 December 1999
Abstract Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter–operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: Ž1. modifying the host cell’s toxicant efflux capacity via a tolC mutation; Ž2. incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; Ž3. changing the reporter element to a different lux system Ž Photorhabdus luminescens., with a broader temperature range; Ž4. using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Ames test; Bioluminescence; Escherichia coli; Genotoxicity testing; lux; recA; SOS response
1. Introduction
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Corresponding author. Tel.: q972-2-658-4192; fax: q972-2658-5559; e-mail: [email protected] 1 Present address: Small Molecule Therapeutics, 11 Deer Park Lane, Suite 116, Monmouth Junction, NJ 08852, USA.
The obvious need for sensitive monitoring of environmental genotoxicants, to warn and protect against mutagenic, carcinogenic and teratogenic effects of pollutants, has led to the development of
1383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 2 3 3 - 8
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numerous methodologies for genotoxicity detection. Assays relevant to human risk assessment often involve mammals, either using direct animal exposure or tissue culture tests. Simpler microbial bioassays, justified by the correlation between genotoxicity of chemicals in bacterial and animal systems w1,2x, allow faster molecular insights and are gaining increasing usage. Of these, the most widely accepted and practiced is the Salmonella mutagenicity assay Žthe ‘‘Ames test’’ w3x, which quantifies reverse, prototrophic mutations in histidine-requiring strains of Salmonella typhimurium. A different approach for the detection of environmental mutagens is based on the activation of the bacterial SOS system. Included in this category are the SOS Chromotest w4x, the Rec–lac test w5x and the umu test w6x. In all of these cases, SOS induction is followed using lacZ as a reporter gene, and chromogenic b-galactosidase activity at a single time point serves as a measure of genotoxicity. Several recent reports have suggested detecting SOS activation by the use of bacterial bioluminescence Ž lux . as a reporter w7–12x, an approach which allows the monitoring of the bacterial response in real-time by simple luminometry. Belkin et al. w7,8x and Vollmer et al. w12x described the use, in Escherichia coli, of either recA or uÕrA promoters with Vibrio fischeri’s luxCDABE. In the Vitotox w assay w10,11x, the same plasmid-borne reporter system was fused to the recN promoter of E. coli and introduced into S. typhimurium. Ptitsyn et al. w9x have used, in E. coli, the fusion of the cda promoter with the luxCDABE
of Photobacterium leiognathi. Elasri and Miller w13x reported, in Pseudomonas aeroginosa, the fusion of its recA promoter to V. fischeri luxCDABE as a means for monitoring UV radiation, as previously shown by Vollmer et al. w12x in E. coli. The systems reported in these studies share several disadvantages, among them the plasmid location of the promoter–lux fusion, and — at least for the E. coli and S. typhimurium constructs — the need to operate at a temperature range dictated by the luminescence enzymes rather than that of the host organism. In a continuation of our previous studies w7,8,12x, we describe in this communication several of the avenues pursued to overcome these difficulties and thus improve genotoxicant detection capacities of bacterial strains carrying recAX
2. Materials and methods 2.1. Bacterial strains and plasmids The different bacterial strains used in this study are listed in Table 1. Construction of the plasmid containing the recAX
Table 1 Bioluminescent bacterial strains used in the present study Strain designation
SOS promoter
Location of lux fusiona
lux originb
tolC c
Host straind
Assay temperature Ž8C.
DPD2794 DPD2797 DPD1718 DPD1714 DPD1709 DPD3063 Sal94
recA recA recA recA recA recA recA
mcp mcp ci ci ci ci mcp
Vf Vf Pl Pl Pl Vf Vf
q y q q q q q
Ec RFM443 w15,41x Ec DE112 w14x Ec DPD1692 Ec DM800 w24x Ec DM803 w24x Ec W3110 w25x St WG49 w16x
26 26 37 37 37 26 26
a
mcp: multi-copy plasmid; ci: chromosomal integration. Vf: V. fischeri; Pl: P. luminescens. c Žq. Wild type; Žy. mutant. d Ec: E. coli; St: S. typhimurium. b
Comments
lexAind
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E. coli strain DPD1707 contains a chromosomally integrated fusion of the E. coli recA promoter region to the Photorhabdus luminescens luxCDABE reporter in host strain JC7623 w17x. This strain was constructed by a previously described method w18x using plasmid pDEW14. Plasmid pDEW14 was constructed by ligating a Pst I–EcoRI fragment containing a recA–luxCDABE fusion from plasmid pDEW65 into plasmid pBRINT.Cm w19x. Previously, plasmid pDEW65 had been made by ligating a fragment containing the recA operator-promoter region into BamHI- and SalI-digested pJT205 w20x. This recA operator-promoter region had been obtained by PCR amplification using pRecALux3 w12x as the template. Strain DPD1707 was used as the donor in the P1 vir-mediated generalized transductions w21x with selection for chloramphenicol resistance to construct E. coli strains DPD1718, DPD1714, and DPD1709. E. coli strain DPD1718 was constructed by using strain DPD1692 ŽLacy Kanamycin-resistant; DuPont Microbial Genetics laboratory collection. as the recipient for the transduction with P1 vir phage grown on strain DPD1707. The recA–lux fusion of strain DPD1718 is integrated into the lacZ locus of E. coli and oriented such that the direction of transcription is the same as that of that of the lacZYA operon. Addition of 30 mgrml nalidixic acid, a DNA gyrase inhibitor that results in potent induction of the SOS response w22x, to an exponentially growing culture of DPD1718 grown at 378C in Vogel–Bonner medium with glucose as the carbon source w23x resulted in a 60-fold increase in bioluminescence 120 min after addition, confirming the transcriptional control of this fusion by the recA promoter. Addition of 1 mM isopropylthio-b-D-galactoside ŽIPTG. to a log phase culture of DPD1718 grown at 378C in the same medium resulted in a sixfold increase in bioluminescence 120 min after addition, suggesting that transcription of the lux genes in this construct was also controlled by the lac operator-promoter. It is unlikely that the chemicals used in this study resulted in activation of transcription from the lac promoter because the magnitude of induction observed with the tested chemicals exceeds that obtained with the lac operon inducer, IPTG. E. coli strains DPD1714 and DPD1709 were constructed by using strains, DM800 F y , metA28, lacY 1 or Z4, thi-1, xyl-5 or -7, galK2, tsx-6 4 w24x
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and DM803 lexAind of DM8004 w24x, respectively, as the recipients in transductions, with P1 vir phage grown on strain DPD1707. lexAind encodes a noncleavable form of the LexA, the repressor of SOS-induced gene expression w22x. E. coli strain DPD3063 contains a chromosomal insertion of the recA promoter region fused to the V. fischeri luxCDABE reporter at the lacZ locus. It was constructed using P1 vir phage grown on strain DPD3062 as the donor and E. coli strain W3110 Fy, prototroph4 w25x as the recipient with selection for Kanamycin resistance. Prior to this, E. coli strain DPD3062 had been made by a previously described method w18x using plasmid pDEW100 that had been constructed by ligation of a 8.6-kbp BamHI–PstI fragment of pRecALux3 w12x into plasmid pBRINT.Km w19x. 2.2. Experimental conditions, luminescence measurement and data analysis Prior to the assay, the bacterial tester strains were grown overnight in LB broth w21x, with shaking at 268C or at 378C, according to the tested strain ŽTable 1.. The cells were then diluted to approximately 10 7 cellsrml and regrown under the same conditions until a cell density of ca. 2 = 10 8 was reached. Kanamycin Ž25 mgrl. was included in the overnight growth medium, to ensure maintenance of the plasmid, but omitted from the regrowth medium due to an inhibitory effect on luminescence ŽS. Belkin, unpublished data.. For the plasmid-bearing strains, repeated controls demonstrated no loss of kanamycin resistance, and hence, no loss of plasmid, during the short assay period. All samples tested were at pH 7.0. A twofold dilution series in LB was prepared in opaque white microtiter plates ŽDynatech, Chantilly, VA, USA., to a final volume of 50 ml in each of the wells. LB broth served as a control. To all wells, 50 ml of the early exponential cell suspension was added, and the plates were incubated in a temperature-controlled Ž268C or 378C, see Table 1. microtiter plate luminometer ŽLucy 1, Anthos Labtech, Salzburg, Austria.. Luminescence values are presented as arbitrary relative light units ŽRLU., or as the ratio of the luminescence of the induced sample to that of the uninduced control Žresponse ratio. as described pre-
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viously w8,29x. Maximal response ratios are the maximal ratios obtained during the course of an experiment. EC 200 is the effective sample concentration causing a twofold increase in the maximal response ratio. As previously described w8,29,30x, EC 200 values were determined by plotting a value known as gamma Ž G . as a function of sample concentration. G is defined as Ž Is y Io .rIo , where Is is the maximal luminescence obtained for the given sample concentration s, and Io is the luminescence of the control at the same time point. When G s 1, sample concentration is equal to EC 200 . All experiments were run in duplicate, and were repeated at least twice Žthree times in most cases. at different dates, using different batches of cells. Standard deviation of duplicates was smaller than 5%.
ing lyophilized V. fischeri preparations purchased from Azur Environmental ŽCarlsbad, CA, USA.. The Microtox w procedure was adapted to microtiter plate format in a manner similar to that published by Blaise et al. w33x.
2.3. Alginate immobilization
3.1. Kinetics of light production and data analysis
Exponentially growing culture aliquots Ž5 ml. were gently mixed with an equal volume of Na-alginate ŽSigma., and added dropwise Ž35–40 mlr drop. into a continuously stirred solution of 0.2 M CaCl 2 . The Ca-alginate beads which were immediately formed were rinsed for several hours in a large volume of fresh CaCl 2 Ž0.2 M. at 48C, and routinely stored under the same conditions.
Three compounds served for the initial comparison of the various E. coli constructs examined in the course of this study: MC, H 2 O 2 and MNNG. To exemplify characteristics of light production by SOS-responsive promoter
2.4. Industrial wastewaters Wastewater samples were neutralized to pH 7, filter-sterilized Ž0.22-mm pore size. and kept frozen until tested. Testing was carried out in microtiter plates as in Section 2.2 above, following a twofold dilution series in LB. Toxicity of the samples was determined by the Microtox w bioassay w31,32x, us-
2.5. Chemicals All chemicals used were analytical grade. Hydrogen peroxide ŽH 2 O 2 ., mitomycin C ŽMC., and 1methyl-3-nitro-1-nitrosoguanidine ŽMNNG. were obtained from Merck, Sigma and Fluca, respectively.
3. Results
X Fig. 1. Genotoxicity determination using the recA
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increase in luminescence, and are characteristic of each tester strainrgenotoxicant couple. When comparing the performance of different strains exposed to the same inducer, lower EC 200 values reflect greater sensitivity and a lower detection threshold of the tested strain; when comparing the response of a single strain to different compounds, lower EC 200 values imply greater genotoxicities of the tested chemicals. 3.2. LexA control of the luminescence response To confirm that the luminescence response measured in the presence of known DNA-damaging agents is indeed SOS-regulated, we have tested chromosomal integrations of the recAX
Fig. 3. A tolC mutation significantly increases sensitivity to MC but not to H 2 O 2 . Strains DPD2797 and DPD2794 are isogenic except for the tolC mutation in the former.
3.3. Inhibiting efflux capacity by a tolC mutation One potential difficulty in assessing the toxic potential of either defined chemicals or unknown samples lies in the permeability barriers posed by the bacterial membrane, hindering access of the toxin to the target macromolecule. It has been demonstrated w14x that the use of an E. coli host strain with a tolC mutation w34x lowered the detection threshold of a sensor strain harboring a grpEX
X
Fig. 2. Induction of recA
The original bioluminescent genotoxicity sensor strains w12x harbored the lux fusion on a multi-copy plasmid, thus enhancing the intensity of the observed response but introducing a potential instability into the maintenance of the extrachromosomal genetic element. Another possible disadvantage of a multicopy plasmid-based fusion is a loss of responsiveness to the regulatory element due to a titration effect of hundreds of fusions containing operatorpromoters on a fixed level of repressor. Strain DPD3063 ŽFig. 1. chromosomally integrates a single
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copy of the same recAX
Fig. 5. The use of P. luminescens lux as a reporter results in a more rapid response. DPD3063 — V. fischeri lux, 268C. DPD1718 — P. luminescens lux, 378C. MC Ž50 mgrl. served as the inducer.
3.5. Expanding temperature range by the use of a different reporter: P. luminescens instead of V. fischeri lux One of the problems often accompanying the use of luminescent E. coli is the necessary compromise in working temperature: the optimal expression temperature of the V. fischeri luminescence enzymes is much lower then the 378C required for ‘‘normal’’ E. coli functions. A solution may be provided by the lux system of P. luminescens w35x, which readily operates at this temperature. As described under Section 2, P. luminescens luxCDABE was therefore fused to the E. coli recA promoter and chromosomally integrated to yield strain DPD1718. When the 378C response of DPD1718 to the three model inducers was compared to that of DPD3063 at 268C,
an advantage in response time was clearly apparent. This is demonstrated in Fig. 5 for MC. When both strains were compared at 268C, DPD1718 still exhibited faster responses than DPD3063, although the differences were less pronounced Žnot shown.. At both temperatures, DPD1718 displayed a higher background luminescence Žnot shown., leading to lower response ratios and thus to a higher detection threshold. The advantage in response times was thus offset by a reduced sensitivity. 3.6. Changing the microbial host from E. coli to S. typhimurium While E. coli is a very convenient test organism, the use of S. typhimurium as a sensor strain seemed attractive due to its general acceptance as a mutagenicity assay organism in the histidine reversion test w3x, as well as to its current use in the Vitotox w assay w10,11x. S. typhimurium strain Sal94 was therefore constructed, containing the same recAX
X
Fig. 4. Chromosomal integration of the recA
3.7. Amenability to polymer immobilization Several options can be envisioned for on-line application of microbial sensor cells to real-time
Y. DaÕidoÕ et al.r Mutation Research 466 (2000) 97–107
Fig. 6. Comparison of S. typhimurium ŽSal94. and E. coli ŽDPD2794. as a host for the recAX
genotoxicity monitoring; one of these includes immobilization in a polymer matrix. In a preliminary attempt to determine if these genotoxicant-responsive strains are amenable to such treatment, tester cultures were encapsulated in alginate and subjected to several maintenance regimes. When stored in a CaCl 2 solution at 48C, the cells remained active for prolonged periods, but at the cost of an increasingly delayed response and reduced luminescence intensity. Fig. 7 summarizes the results of one such experiment carried out with strain DPD2797 Ž tolC, V. fischeri lux fusion on a multi-copy plasmid.. Over the first month, maximal luminescence values and response ratios decreased only marginally, while the lag period doubled from 1 to 2 h. EC 200 values were not significantly affected during this initial storage time Žnot shown.. During the second month, all three parameters deteriorated, and after 60 days, the response ratio elicited by 100 mgrl MC decreased to approximately 1.8. After the third month, although the cells were still luminescent, the reaction to MC was very weak and delayed by over 5 h.
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halomethanes. At least for the compounds in Table 2, two strains stand out with the lowest detection levels: DPD3063 and DPD2797. While these strains exhibited positive responses to several Ames-negative compounds, except for one case, all of these chemicals were previously recorded as potential genotoxicants by at least one other established bioassay. For the one exception in Table 2, the Amesnegative 2,3 dichlorophenol, no conclusive data could be found to prove or disprove its genotoxic potential. There were no false-negatives among the tested chemicals; furthermore, numerous other Ames-negative compounds, with no recorded genotoxic activity, did not induce any of the recAX
3.8. Detection threshold for other chemicals Table 2 displays EC 200 data for an expanded list of chemicals, most of them known or suspected genotoxicants w36,37x, obtained by the use of selected strains from among those described in Table 1. In addition to the three model compounds used in the examples presented above, Table 2 contains representatives of two groups of environmentally significant chemicals: phenol and some derivatives, and
Fig. 7. Response to MC Ž100 mgrl. of alginate-embedded cells of strain DPD2797 as a function of storage time at 48C.
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104 Table 2 EC200 values for selected chemicals Compound MC MNNG H 2 O2 Phenol 2,4,6 Trichlorophenol 4-Nitrophenol 2,3 Dichlorophenol Pentachlorophenol Chlorodibromomethane Bromodichloromethane Chloroform
CAS number 50-07-7 70-25-7 7722-84-1 108-95-2 88-06-2 100-02-7 576-24-9 87-86-5 124-48-1 75-24-4 67-66-3
Tester straina DPD3063 DPD2797 DPD3063 Sal94 DPD2822 Sal94 DPD3063 DPD3063 DPD3063rDPD2797 DPD2797 DPD3063
b Žmgrl. EC 200 y4
y5
10 " 8 = 10 0.05 " 0.025 0.8 " 0.5 16 10 " 3.5 0.25 50 0.008 20 " 8 100 300
Ames test c
Other genotoxicity tests d
q q q y y y y y y q y
ND ND ND q q q ND q q q q
a
Strain for which lowest EC 200 values were obtained. EC 200 values for the strain in the previous column. Data represent average" standard error Žfor three or more independent experiments. or average only Žonly two independent experiments.. Each independent experiment was carried out in duplicate. c Data from Zeiger w37x. Žq. Positive response; Žy. negative response. d Data from Zeiger w37x. Žq. positive response in at least one of the following assays: cultured Chinese hamster in vitro chromosome aberration, in vitro Chinese hamster sister chromatid exchange, mouse lymphoma cell mutation, or Drosophila sex-linked recessive lethality; ND — no data. b
in 10 consecutive days of February and March 1994, from both the influent and the effluent of a biological Žactivated sludge. treatment plant of the wastewaters of a chemical manufacturing facility. The influents were characterized by an average dissolved organic load of 640 mg Crl, a total dissolved solids content of 1050 mgrl, and a relatively high Microtox w w31,32x toxicity ŽEC 50 : 1.5%.. The effluents, in contrast, did not display any Microtox w toxicity, and while their mineral contact remained unchanged, the dissolved organic carbon was reduced, on the aver-
age, to 18 mgrl. Fig. 8 presents the induction ratios of strain DPD2794 in response to this sample series Žall at 10% concentration.. All crude wastewater samples displayed a variable but clear SOS inductive effect, thus indicating a potential genotoxicity; this activity, however, was completely eliminated by the biological activity in the treatment plant. At least by our assay, the effluents did not contain the genotoxic hazards present in the raw influents, which were either degraded or neutralized during the treatment process.
4. Discussion
Fig. 8. Response of strain DPD2794 to chemical industrial wastewaters collected at 10 consecutive days in 1994, before and after treatment in a biological activated sludge reactor. All samples were tested at 10% strength.
While the need for sensitive, rapid, and cost-effective means for eco-genotoxicology monitoring is obvious, it is also clear that no single test can provide a definite and comprehensive picture of the genotoxic hazards inherent in a sample. As in acute toxicity bioassays, therefore, a ‘‘battery approach’’ may be needed for a complete coverage of all potential risks, although this may be hampered by cost considerations. Out of presently available methodologies, those based on reporter gene fusions to SOS promoters appear, in principle, to be highly cost-effective, at least for screening purposes. Though SOS
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activation in itself does not imply mutagenicity, the two activities are certainly correlated w4x. The SOS Chromotest w4x and umu test w6x both utilize this phenomenon: following an incubation period of a fixed length, a colorimetric assay is performed to quantify the b-galactosidase activity driven by SOS induction. This communication describes developments in the use of a different reporter for SOS activation: microbial bioluminescence Ž lux . genes. Past reports w9–12x have described different pathways in the choice of the three major system components: SOS operator-promoter, lux reporter and host organism. The end result, however, was similar in all cases: plasmid-bearing bacterial strains capable of sensitive bioluminescent reporting on the presence of DNAdamaging agents. US and European patents pertaining to the use of lux reporter technology in this manner have recently been granted w38,39x, and one of the tests described — Vitotox w — has recently become commercially available. Our original report w12x described the use, in E. coli, of either recA or uÕrA promoters with V. fischeri’s luxCDABE. Strain DPD2794, bearing the recAX
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be used in the future to endow the tester strains with a broader spectrum of enhanced sensitivity. 4.2. Change of reporter genes from V. fischeri to P. luminescens lux The main purpose of this step was to expand the temperature range of the reporter proteins into that optimal for E. coli. The higher temperature caused a faster induction of the SOS system and thus of the lux operon, leading to a faster luminescence response; this advantage, however, was offset by much higher background luminescence, which significantly affected the sensitivity of the system. 4.3. Integration of the promoter
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a single chromosomal integration of the recAX
w10x
Acknowledgements
w11x
The authors wish to thank Mary Jane Reeve and Hemant Purhoit for their excellent assistance, and R. Armon for S. typhimurium strain WG49. Research in the laboratory of S. Belkin was supported, in part, by the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, by the Interuniversity Ecology Fund of the Keren Kayemeth LeIsrael, and by the Ministry of Science and the Arts under infrastructure grant no. 13139-1-98.
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ity test systems — where they stand today, in: P.G. Wells, K. Lee, C. Blaise ŽEds.., Microscale Testing in Aquatic Toxicology: Advances, Techniques, and Practice, CRC Press, Boca Raton, FL, 1998, pp. 185–199. C. Blaise, R. Forghani, R. Legault, J. Guzzo, M.S. Dubow, A bacterial toxicity assay performed with microplates, microluminometry and Microtox reagent, Biotechniques 16 Ž1994. 932–937. J.A. Fralick, Evidence that TolC is required for functioning of the MarrAcrAB efflux pump of Escherichia coli, J. Bacteriol. 178 Ž1996. 5803–5805. R. Szittner, E. Meighen, Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium, J. Biol. Chem. 265 Ž1990. 16581– 16587. L.S. Gold, T.H. Slone, N.B. Manley, G.B. Garfinkel, L. Rohrbach, B.N. Ames, Carcinogenic potency database, in: L.S. Gold, E. Zeiger ŽEds.., Handbook of Carcinogenic Potency and Genotoxicity Databases, CRC Press, Boca Raton, FL, 1997, pp. 1–605. E. Zeiger, Genotoxicity database, in: L.S. Gold, E. Zeiger ŽEds.., Handbook of Carcinogenic Potency and Genotoxicity Databases, CRC Press, Boca Raton, FL, 1997, pp. 687–729. R.A. LaRossa, W.M. Majarian, T.K. Van Dyk, Highly sensitive method for detecting environmental insults, US patent 5,683,868, 1997. D. Van der Lelie, B. Borremans, A. Provoost, L. Regniers, L. Verschaeve, Recombinant nucleic acid sequences and methods for determining both genotoxicity and mutagenicity of a sample and the kinetics of the genotoxicity, EU patent EP907748A1, 1999. J.H. Min, E.J. Kim, R.A. LaRossa, M.B. Gu, Distinct responses of a recA
Improved bacterial SOS promoter
DiÕision of EnÕironmental Sciences, the Fredy and Nadine Herrmann Graduate School of Applied Science, The Hebrew UniÕersity of Jerusalem, Jerusalem 91904, Israel b DuPont Central Life Sciences, P.O. Box 80173, Wilmington, DE 19880-0173, USA c Swarthmore College, Swarthmore, PA 19081-1397, USA Received 15 September 1999; received in revised form 8 December 1999; accepted 14 December 1999
Abstract Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter–operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: Ž1. modifying the host cell’s toxicant efflux capacity via a tolC mutation; Ž2. incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; Ž3. changing the reporter element to a different lux system Ž Photorhabdus luminescens., with a broader temperature range; Ž4. using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Ames test; Bioluminescence; Escherichia coli; Genotoxicity testing; lux; recA; SOS response
1. Introduction
)
Corresponding author. Tel.: q972-2-658-4192; fax: q972-2658-5559; e-mail: [email protected] 1 Present address: Small Molecule Therapeutics, 11 Deer Park Lane, Suite 116, Monmouth Junction, NJ 08852, USA.
The obvious need for sensitive monitoring of environmental genotoxicants, to warn and protect against mutagenic, carcinogenic and teratogenic effects of pollutants, has led to the development of
1383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 2 3 3 - 8
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numerous methodologies for genotoxicity detection. Assays relevant to human risk assessment often involve mammals, either using direct animal exposure or tissue culture tests. Simpler microbial bioassays, justified by the correlation between genotoxicity of chemicals in bacterial and animal systems w1,2x, allow faster molecular insights and are gaining increasing usage. Of these, the most widely accepted and practiced is the Salmonella mutagenicity assay Žthe ‘‘Ames test’’ w3x, which quantifies reverse, prototrophic mutations in histidine-requiring strains of Salmonella typhimurium. A different approach for the detection of environmental mutagens is based on the activation of the bacterial SOS system. Included in this category are the SOS Chromotest w4x, the Rec–lac test w5x and the umu test w6x. In all of these cases, SOS induction is followed using lacZ as a reporter gene, and chromogenic b-galactosidase activity at a single time point serves as a measure of genotoxicity. Several recent reports have suggested detecting SOS activation by the use of bacterial bioluminescence Ž lux . as a reporter w7–12x, an approach which allows the monitoring of the bacterial response in real-time by simple luminometry. Belkin et al. w7,8x and Vollmer et al. w12x described the use, in Escherichia coli, of either recA or uÕrA promoters with Vibrio fischeri’s luxCDABE. In the Vitotox w assay w10,11x, the same plasmid-borne reporter system was fused to the recN promoter of E. coli and introduced into S. typhimurium. Ptitsyn et al. w9x have used, in E. coli, the fusion of the cda promoter with the luxCDABE
of Photobacterium leiognathi. Elasri and Miller w13x reported, in Pseudomonas aeroginosa, the fusion of its recA promoter to V. fischeri luxCDABE as a means for monitoring UV radiation, as previously shown by Vollmer et al. w12x in E. coli. The systems reported in these studies share several disadvantages, among them the plasmid location of the promoter–lux fusion, and — at least for the E. coli and S. typhimurium constructs — the need to operate at a temperature range dictated by the luminescence enzymes rather than that of the host organism. In a continuation of our previous studies w7,8,12x, we describe in this communication several of the avenues pursued to overcome these difficulties and thus improve genotoxicant detection capacities of bacterial strains carrying recAX
2. Materials and methods 2.1. Bacterial strains and plasmids The different bacterial strains used in this study are listed in Table 1. Construction of the plasmid containing the recAX
Table 1 Bioluminescent bacterial strains used in the present study Strain designation
SOS promoter
Location of lux fusiona
lux originb
tolC c
Host straind
Assay temperature Ž8C.
DPD2794 DPD2797 DPD1718 DPD1714 DPD1709 DPD3063 Sal94
recA recA recA recA recA recA recA
mcp mcp ci ci ci ci mcp
Vf Vf Pl Pl Pl Vf Vf
q y q q q q q
Ec RFM443 w15,41x Ec DE112 w14x Ec DPD1692 Ec DM800 w24x Ec DM803 w24x Ec W3110 w25x St WG49 w16x
26 26 37 37 37 26 26
a
mcp: multi-copy plasmid; ci: chromosomal integration. Vf: V. fischeri; Pl: P. luminescens. c Žq. Wild type; Žy. mutant. d Ec: E. coli; St: S. typhimurium. b
Comments
lexAind
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E. coli strain DPD1707 contains a chromosomally integrated fusion of the E. coli recA promoter region to the Photorhabdus luminescens luxCDABE reporter in host strain JC7623 w17x. This strain was constructed by a previously described method w18x using plasmid pDEW14. Plasmid pDEW14 was constructed by ligating a Pst I–EcoRI fragment containing a recA–luxCDABE fusion from plasmid pDEW65 into plasmid pBRINT.Cm w19x. Previously, plasmid pDEW65 had been made by ligating a fragment containing the recA operator-promoter region into BamHI- and SalI-digested pJT205 w20x. This recA operator-promoter region had been obtained by PCR amplification using pRecALux3 w12x as the template. Strain DPD1707 was used as the donor in the P1 vir-mediated generalized transductions w21x with selection for chloramphenicol resistance to construct E. coli strains DPD1718, DPD1714, and DPD1709. E. coli strain DPD1718 was constructed by using strain DPD1692 ŽLacy Kanamycin-resistant; DuPont Microbial Genetics laboratory collection. as the recipient for the transduction with P1 vir phage grown on strain DPD1707. The recA–lux fusion of strain DPD1718 is integrated into the lacZ locus of E. coli and oriented such that the direction of transcription is the same as that of that of the lacZYA operon. Addition of 30 mgrml nalidixic acid, a DNA gyrase inhibitor that results in potent induction of the SOS response w22x, to an exponentially growing culture of DPD1718 grown at 378C in Vogel–Bonner medium with glucose as the carbon source w23x resulted in a 60-fold increase in bioluminescence 120 min after addition, confirming the transcriptional control of this fusion by the recA promoter. Addition of 1 mM isopropylthio-b-D-galactoside ŽIPTG. to a log phase culture of DPD1718 grown at 378C in the same medium resulted in a sixfold increase in bioluminescence 120 min after addition, suggesting that transcription of the lux genes in this construct was also controlled by the lac operator-promoter. It is unlikely that the chemicals used in this study resulted in activation of transcription from the lac promoter because the magnitude of induction observed with the tested chemicals exceeds that obtained with the lac operon inducer, IPTG. E. coli strains DPD1714 and DPD1709 were constructed by using strains, DM800 F y , metA28, lacY 1 or Z4, thi-1, xyl-5 or -7, galK2, tsx-6 4 w24x
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and DM803 lexAind of DM8004 w24x, respectively, as the recipients in transductions, with P1 vir phage grown on strain DPD1707. lexAind encodes a noncleavable form of the LexA, the repressor of SOS-induced gene expression w22x. E. coli strain DPD3063 contains a chromosomal insertion of the recA promoter region fused to the V. fischeri luxCDABE reporter at the lacZ locus. It was constructed using P1 vir phage grown on strain DPD3062 as the donor and E. coli strain W3110 Fy, prototroph4 w25x as the recipient with selection for Kanamycin resistance. Prior to this, E. coli strain DPD3062 had been made by a previously described method w18x using plasmid pDEW100 that had been constructed by ligation of a 8.6-kbp BamHI–PstI fragment of pRecALux3 w12x into plasmid pBRINT.Km w19x. 2.2. Experimental conditions, luminescence measurement and data analysis Prior to the assay, the bacterial tester strains were grown overnight in LB broth w21x, with shaking at 268C or at 378C, according to the tested strain ŽTable 1.. The cells were then diluted to approximately 10 7 cellsrml and regrown under the same conditions until a cell density of ca. 2 = 10 8 was reached. Kanamycin Ž25 mgrl. was included in the overnight growth medium, to ensure maintenance of the plasmid, but omitted from the regrowth medium due to an inhibitory effect on luminescence ŽS. Belkin, unpublished data.. For the plasmid-bearing strains, repeated controls demonstrated no loss of kanamycin resistance, and hence, no loss of plasmid, during the short assay period. All samples tested were at pH 7.0. A twofold dilution series in LB was prepared in opaque white microtiter plates ŽDynatech, Chantilly, VA, USA., to a final volume of 50 ml in each of the wells. LB broth served as a control. To all wells, 50 ml of the early exponential cell suspension was added, and the plates were incubated in a temperature-controlled Ž268C or 378C, see Table 1. microtiter plate luminometer ŽLucy 1, Anthos Labtech, Salzburg, Austria.. Luminescence values are presented as arbitrary relative light units ŽRLU., or as the ratio of the luminescence of the induced sample to that of the uninduced control Žresponse ratio. as described pre-
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viously w8,29x. Maximal response ratios are the maximal ratios obtained during the course of an experiment. EC 200 is the effective sample concentration causing a twofold increase in the maximal response ratio. As previously described w8,29,30x, EC 200 values were determined by plotting a value known as gamma Ž G . as a function of sample concentration. G is defined as Ž Is y Io .rIo , where Is is the maximal luminescence obtained for the given sample concentration s, and Io is the luminescence of the control at the same time point. When G s 1, sample concentration is equal to EC 200 . All experiments were run in duplicate, and were repeated at least twice Žthree times in most cases. at different dates, using different batches of cells. Standard deviation of duplicates was smaller than 5%.
ing lyophilized V. fischeri preparations purchased from Azur Environmental ŽCarlsbad, CA, USA.. The Microtox w procedure was adapted to microtiter plate format in a manner similar to that published by Blaise et al. w33x.
2.3. Alginate immobilization
3.1. Kinetics of light production and data analysis
Exponentially growing culture aliquots Ž5 ml. were gently mixed with an equal volume of Na-alginate ŽSigma., and added dropwise Ž35–40 mlr drop. into a continuously stirred solution of 0.2 M CaCl 2 . The Ca-alginate beads which were immediately formed were rinsed for several hours in a large volume of fresh CaCl 2 Ž0.2 M. at 48C, and routinely stored under the same conditions.
Three compounds served for the initial comparison of the various E. coli constructs examined in the course of this study: MC, H 2 O 2 and MNNG. To exemplify characteristics of light production by SOS-responsive promoter
2.4. Industrial wastewaters Wastewater samples were neutralized to pH 7, filter-sterilized Ž0.22-mm pore size. and kept frozen until tested. Testing was carried out in microtiter plates as in Section 2.2 above, following a twofold dilution series in LB. Toxicity of the samples was determined by the Microtox w bioassay w31,32x, us-
2.5. Chemicals All chemicals used were analytical grade. Hydrogen peroxide ŽH 2 O 2 ., mitomycin C ŽMC., and 1methyl-3-nitro-1-nitrosoguanidine ŽMNNG. were obtained from Merck, Sigma and Fluca, respectively.
3. Results
X Fig. 1. Genotoxicity determination using the recA
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increase in luminescence, and are characteristic of each tester strainrgenotoxicant couple. When comparing the performance of different strains exposed to the same inducer, lower EC 200 values reflect greater sensitivity and a lower detection threshold of the tested strain; when comparing the response of a single strain to different compounds, lower EC 200 values imply greater genotoxicities of the tested chemicals. 3.2. LexA control of the luminescence response To confirm that the luminescence response measured in the presence of known DNA-damaging agents is indeed SOS-regulated, we have tested chromosomal integrations of the recAX
Fig. 3. A tolC mutation significantly increases sensitivity to MC but not to H 2 O 2 . Strains DPD2797 and DPD2794 are isogenic except for the tolC mutation in the former.
3.3. Inhibiting efflux capacity by a tolC mutation One potential difficulty in assessing the toxic potential of either defined chemicals or unknown samples lies in the permeability barriers posed by the bacterial membrane, hindering access of the toxin to the target macromolecule. It has been demonstrated w14x that the use of an E. coli host strain with a tolC mutation w34x lowered the detection threshold of a sensor strain harboring a grpEX
X
Fig. 2. Induction of recA
The original bioluminescent genotoxicity sensor strains w12x harbored the lux fusion on a multi-copy plasmid, thus enhancing the intensity of the observed response but introducing a potential instability into the maintenance of the extrachromosomal genetic element. Another possible disadvantage of a multicopy plasmid-based fusion is a loss of responsiveness to the regulatory element due to a titration effect of hundreds of fusions containing operatorpromoters on a fixed level of repressor. Strain DPD3063 ŽFig. 1. chromosomally integrates a single
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copy of the same recAX
Fig. 5. The use of P. luminescens lux as a reporter results in a more rapid response. DPD3063 — V. fischeri lux, 268C. DPD1718 — P. luminescens lux, 378C. MC Ž50 mgrl. served as the inducer.
3.5. Expanding temperature range by the use of a different reporter: P. luminescens instead of V. fischeri lux One of the problems often accompanying the use of luminescent E. coli is the necessary compromise in working temperature: the optimal expression temperature of the V. fischeri luminescence enzymes is much lower then the 378C required for ‘‘normal’’ E. coli functions. A solution may be provided by the lux system of P. luminescens w35x, which readily operates at this temperature. As described under Section 2, P. luminescens luxCDABE was therefore fused to the E. coli recA promoter and chromosomally integrated to yield strain DPD1718. When the 378C response of DPD1718 to the three model inducers was compared to that of DPD3063 at 268C,
an advantage in response time was clearly apparent. This is demonstrated in Fig. 5 for MC. When both strains were compared at 268C, DPD1718 still exhibited faster responses than DPD3063, although the differences were less pronounced Žnot shown.. At both temperatures, DPD1718 displayed a higher background luminescence Žnot shown., leading to lower response ratios and thus to a higher detection threshold. The advantage in response times was thus offset by a reduced sensitivity. 3.6. Changing the microbial host from E. coli to S. typhimurium While E. coli is a very convenient test organism, the use of S. typhimurium as a sensor strain seemed attractive due to its general acceptance as a mutagenicity assay organism in the histidine reversion test w3x, as well as to its current use in the Vitotox w assay w10,11x. S. typhimurium strain Sal94 was therefore constructed, containing the same recAX
X
Fig. 4. Chromosomal integration of the recA
3.7. Amenability to polymer immobilization Several options can be envisioned for on-line application of microbial sensor cells to real-time
Y. DaÕidoÕ et al.r Mutation Research 466 (2000) 97–107
Fig. 6. Comparison of S. typhimurium ŽSal94. and E. coli ŽDPD2794. as a host for the recAX
genotoxicity monitoring; one of these includes immobilization in a polymer matrix. In a preliminary attempt to determine if these genotoxicant-responsive strains are amenable to such treatment, tester cultures were encapsulated in alginate and subjected to several maintenance regimes. When stored in a CaCl 2 solution at 48C, the cells remained active for prolonged periods, but at the cost of an increasingly delayed response and reduced luminescence intensity. Fig. 7 summarizes the results of one such experiment carried out with strain DPD2797 Ž tolC, V. fischeri lux fusion on a multi-copy plasmid.. Over the first month, maximal luminescence values and response ratios decreased only marginally, while the lag period doubled from 1 to 2 h. EC 200 values were not significantly affected during this initial storage time Žnot shown.. During the second month, all three parameters deteriorated, and after 60 days, the response ratio elicited by 100 mgrl MC decreased to approximately 1.8. After the third month, although the cells were still luminescent, the reaction to MC was very weak and delayed by over 5 h.
103
halomethanes. At least for the compounds in Table 2, two strains stand out with the lowest detection levels: DPD3063 and DPD2797. While these strains exhibited positive responses to several Ames-negative compounds, except for one case, all of these chemicals were previously recorded as potential genotoxicants by at least one other established bioassay. For the one exception in Table 2, the Amesnegative 2,3 dichlorophenol, no conclusive data could be found to prove or disprove its genotoxic potential. There were no false-negatives among the tested chemicals; furthermore, numerous other Ames-negative compounds, with no recorded genotoxic activity, did not induce any of the recAX
3.8. Detection threshold for other chemicals Table 2 displays EC 200 data for an expanded list of chemicals, most of them known or suspected genotoxicants w36,37x, obtained by the use of selected strains from among those described in Table 1. In addition to the three model compounds used in the examples presented above, Table 2 contains representatives of two groups of environmentally significant chemicals: phenol and some derivatives, and
Fig. 7. Response to MC Ž100 mgrl. of alginate-embedded cells of strain DPD2797 as a function of storage time at 48C.
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104 Table 2 EC200 values for selected chemicals Compound MC MNNG H 2 O2 Phenol 2,4,6 Trichlorophenol 4-Nitrophenol 2,3 Dichlorophenol Pentachlorophenol Chlorodibromomethane Bromodichloromethane Chloroform
CAS number 50-07-7 70-25-7 7722-84-1 108-95-2 88-06-2 100-02-7 576-24-9 87-86-5 124-48-1 75-24-4 67-66-3
Tester straina DPD3063 DPD2797 DPD3063 Sal94 DPD2822 Sal94 DPD3063 DPD3063 DPD3063rDPD2797 DPD2797 DPD3063
b Žmgrl. EC 200 y4
y5
10 " 8 = 10 0.05 " 0.025 0.8 " 0.5 16 10 " 3.5 0.25 50 0.008 20 " 8 100 300
Ames test c
Other genotoxicity tests d
q q q y y y y y y q y
ND ND ND q q q ND q q q q
a
Strain for which lowest EC 200 values were obtained. EC 200 values for the strain in the previous column. Data represent average" standard error Žfor three or more independent experiments. or average only Žonly two independent experiments.. Each independent experiment was carried out in duplicate. c Data from Zeiger w37x. Žq. Positive response; Žy. negative response. d Data from Zeiger w37x. Žq. positive response in at least one of the following assays: cultured Chinese hamster in vitro chromosome aberration, in vitro Chinese hamster sister chromatid exchange, mouse lymphoma cell mutation, or Drosophila sex-linked recessive lethality; ND — no data. b
in 10 consecutive days of February and March 1994, from both the influent and the effluent of a biological Žactivated sludge. treatment plant of the wastewaters of a chemical manufacturing facility. The influents were characterized by an average dissolved organic load of 640 mg Crl, a total dissolved solids content of 1050 mgrl, and a relatively high Microtox w w31,32x toxicity ŽEC 50 : 1.5%.. The effluents, in contrast, did not display any Microtox w toxicity, and while their mineral contact remained unchanged, the dissolved organic carbon was reduced, on the aver-
age, to 18 mgrl. Fig. 8 presents the induction ratios of strain DPD2794 in response to this sample series Žall at 10% concentration.. All crude wastewater samples displayed a variable but clear SOS inductive effect, thus indicating a potential genotoxicity; this activity, however, was completely eliminated by the biological activity in the treatment plant. At least by our assay, the effluents did not contain the genotoxic hazards present in the raw influents, which were either degraded or neutralized during the treatment process.
4. Discussion
Fig. 8. Response of strain DPD2794 to chemical industrial wastewaters collected at 10 consecutive days in 1994, before and after treatment in a biological activated sludge reactor. All samples were tested at 10% strength.
While the need for sensitive, rapid, and cost-effective means for eco-genotoxicology monitoring is obvious, it is also clear that no single test can provide a definite and comprehensive picture of the genotoxic hazards inherent in a sample. As in acute toxicity bioassays, therefore, a ‘‘battery approach’’ may be needed for a complete coverage of all potential risks, although this may be hampered by cost considerations. Out of presently available methodologies, those based on reporter gene fusions to SOS promoters appear, in principle, to be highly cost-effective, at least for screening purposes. Though SOS
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activation in itself does not imply mutagenicity, the two activities are certainly correlated w4x. The SOS Chromotest w4x and umu test w6x both utilize this phenomenon: following an incubation period of a fixed length, a colorimetric assay is performed to quantify the b-galactosidase activity driven by SOS induction. This communication describes developments in the use of a different reporter for SOS activation: microbial bioluminescence Ž lux . genes. Past reports w9–12x have described different pathways in the choice of the three major system components: SOS operator-promoter, lux reporter and host organism. The end result, however, was similar in all cases: plasmid-bearing bacterial strains capable of sensitive bioluminescent reporting on the presence of DNAdamaging agents. US and European patents pertaining to the use of lux reporter technology in this manner have recently been granted w38,39x, and one of the tests described — Vitotox w — has recently become commercially available. Our original report w12x described the use, in E. coli, of either recA or uÕrA promoters with V. fischeri’s luxCDABE. Strain DPD2794, bearing the recAX
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be used in the future to endow the tester strains with a broader spectrum of enhanced sensitivity. 4.2. Change of reporter genes from V. fischeri to P. luminescens lux The main purpose of this step was to expand the temperature range of the reporter proteins into that optimal for E. coli. The higher temperature caused a faster induction of the SOS system and thus of the lux operon, leading to a faster luminescence response; this advantage, however, was offset by much higher background luminescence, which significantly affected the sensitivity of the system. 4.3. Integration of the promoter
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a single chromosomal integration of the recAX
w10x
Acknowledgements
w11x
The authors wish to thank Mary Jane Reeve and Hemant Purhoit for their excellent assistance, and R. Armon for S. typhimurium strain WG49. Research in the laboratory of S. Belkin was supported, in part, by the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, by the Interuniversity Ecology Fund of the Keren Kayemeth LeIsrael, and by the Ministry of Science and the Arts under infrastructure grant no. 13139-1-98.
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