A Panel of Bioluminescent Biosensors for Characterization of

Chapter 12

A Panel of Bioluminescent Biosensors for Characterization of Chemically-Induced Bacterial Stress Responses 1,2

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T . K . V a n Dyk , D . R . Smulski , D . A. Elsemore , R . A. LaRossa , and R . W . Morgan Downloaded by STANFORD UNIV GREEN LIBR on September 22, 2012 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0762.ch012

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Central Research and Development Department, DuPont Company, P . O . Box 80173, Willmington, DE 19880-0173 Department of Animal and Food Sciences, University of Delaware, Newark, DE 19717-1303 2

The pattern o f stress responses induced by chemical treatment can characterize types of damage sustained by the cell. Such analyses, which supply insight into the molecular mechanisms of toxicity, may be applied to environmental monitoring. A panel of six whole cell biosensors containing selected stress-responsive Escherichia coli promoters fused to the Photorhabdus luminescens luxCDABE reporter was assembled. These fusions were: recA-lux (in the SOS regulon), grpE-lux (in the heat shock regulon), katG-lux (in the OxyR regulon), inaA-lux (internal acidification responsive and in the Sox and Mar regulons), yciG-lux (in the σ -dependent stress response regulon) and o513-lux (a σ -independent, stationary phase inducible fusion). Two versions of this panel were constructed; one set of strains was in a tolC background and the other harbored a mutation in tolC that inactivates an outer membrane channel for efflux pumps. The responses of this collection were found to be biologically appropriate when stressed by five chemicals with well -defined modes of action, H O , nalidixic acid, ethanol, sodium salicylate, and methyl viologen. Treatment o f this panel with several potential a n i m a l feed contaminants, 2,4dichlorophenoxyacetic acid, cadmium chloride, aluminum chloride, and aflatoxin B1 also yielded characteristic stress fingerprints. s

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© 2000 American Chemical Society

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In Chemical and Biological Sensors for Environmental Monitoring; Mulchandani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Downloaded by STANFORD UNIV GREEN LIBR on September 22, 2012 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0762.ch012

Introduction The responses of an organism to chemical insult can reveal molecular modes of toxicity because these responses typically include repair mechanisms specific for the damage incurred. Many such damage induced stress responses are common throughout biology. For example, the heat shock response, which results in elevated expression of proteins such as molecular chaperones and proteases, is induced in essentially all organisms by stresses that result i n the accumulation o f non-native proteins in the cell (1). D N A damage induced stress responses are also prevalent in biological systems. In E. coli, the SOS response, which results in increased levels of proteins specific for repair of D N A lesions, is induced by D N A damage (2). Consequently, induction of the SOS response i n E. coli serves as a biological indicator of D N A damage caused by chemicals and other treatments. Accordingly, induction of the SOS response in E. coli correlates well with carcinogenicity in mammals (3). Thus, the bacterial heat shock, SOS and other stress responses may be good models to detect and discover modes of chemical toxicity. Many damage inducible responses of microbial cells are regulated at the transcriptional level. A s a result, an approach to rapidly gain insight into the cellular stresses sustained by chemical treatment is genetic fusions of reporter genes to stressinducible promoters. In bacterial systems, a panel of strains utilizing the lacZ reporter has been demonstrated to yield specific responses to various chemical stresses (4). Another sensitive reporter o f changes i n transcription is that o f bacterial bioluminescence encoded by luxCDABE operons. Use of the five gene lux reporter system allows facile monitoring of gene expression because all components necessary for light production are present in the cell. Panels of E. coli strains containing fusions of stress-responsive promoters to the Vibrio fischeri luxCDABE reporter have been described (5) and applied to characterizing the stresses induced by pollutant molecules (6), wastewater (7,8), antibacterial treatments (9,10% and antibiotics (1113). Here, a new panel of six stress-responsive E. coli promoters fused to the Photorhabdus luminescens luxCDABE is described. The P. luminescens lux operon encodes proteins that are more thermostable than those encoded by the V. fischeri lux operon (14). Thus, these biosensor strains were used at optimal temperatures for E. coli growth and, hence, optimal kinetics o f stress response detection were possible. The six members of the panel were chosen to represent a range of stress responses such that different chemical treatments were expected to result in different patterns of induced gene expression. The recA gene is both a regulator and member of the SOS regulon (2). Thus, the transcriptional fusion of the recA promoter to the lux reporter responded to D N A damage. Likewise, because grpE is in the heat shock regulon controlled by 0 (15), the grpE-lux fusion responded to stresses that induce this protein-damage responsive regulon. The katG gene is a member of the OxyR regulon (16) thus, the katG-lux fusion responded to oxidative damage mediated by hydrogen peroxide. A role for the stationary phase sigma factor, 0 , has also been demonstrated in expression of katG in E. coli (17). The inaA gene is induced by treatments that result in internal acidification (18,19) and is controlled by the regulator of the multiple antibiotic stress response circuit, M a r A , the regulator of the superoxide anion stress response circuit, SoxS, and by a protein related to both M a r A and SoxS, Rob (20,21). Accordingly, the inaA-lux fusion responded to stresses that induced these 3 2

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regulatory circuits. Expression of the yciG gene is under control of 0 (21), and thus, the yciG-lux fusion reported on the activation o f the 0 -dependent stress response. Finally, expression of a lux fusion to an otherwise uncharacterized open reading frame o513 is induced by treatment with the herbicide sulfometuron methyl (21). The expression o f this lux fusion was also observed to be highly induced as the culture aged, suggesting stationary phase regulation (T. K . V a n D y k , unpublished observations); however, expression is not controlled by 0 (21). To demonstrate the utility o f this panel, the characteristic stress fingerprints generated by treatment with several toxic substances that may be found as contaminants o f animal feed were determined. These included the commonly used herbicide, 2,4-diehlorophenoxyacetie acid (2,4-D),which is an EPA-regulated hazardous substance and i f present i n feed may result i n accumulation o f this molecule within the animal (22). Also tested were two metal salts that have adverse effects on animal performance when at sufficiently high concentrations in the feed; aluminum salts are toxic i n the range o f 500 to 2000 parts per million (ppm) i n poultry feed, and cadmium salts are toxic in the range o f 12 to 40 ppm for chickens and turkeys (23). Finally, aflatoxin B l , a particularly toxic mycotoxin that can be produced following fungal contamination of animal feed components, was tested. Aflatoxin B l has adverse effects on poultry production when present at about 1 ppm in feed (24-26); the lethal dose for broiler chicks is 70 ppm in the feed (27). s


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Materials and Methods

Bacterial Whole Cell Biosensor Strains Table I lists two versions of the panel of stress-responsive strains. One set carries a tolC mutation, rendering each strain hypersensitive to a variety o f organic molecules; the other set is tolCf'. A s detailed in the following paragraphs, these strains contain either a chromosomal integrant of the lux fusion or a plasmid-borne lux fusion. Furthermore, two o f the strains with plasmid-borne lux fusions carry a chromosomal mutation i n the pcnB gene resulting i n a reduced copy number o f the plasmid. These modifications fine-tuned the basal level o f bioluminescence from unstressed cells to the same general range. E. coli strain DPD2227 contains a plasmid harboring the E. coli katG promoter fused to the P. luminescens luxCDABE in a host strain containing a pcnB mutation that resulted i n reduced copy number o f the plasmid. The plasmid i n this strain, pDEW228 was constructed by digesting plasmid pKatGLux2 (28) with restriction endonuclease Sail, filling in the ends with the Klenow fragment of D N A polymerase I, isolating the 650 basepair fragment containing the E. coli katG promoter region, and ligating it into pDEW201 (29) that had been digested with the restriction endonuclease Smal. The orientation of the katG promoter driving expression o f the luxCDABE operon was confirmed by digestion with the restriction endonuclease Hindlll and by the H202-inducible bioluminescence increases i n strains containing pDEW228. E. coli strain DPD2200 contained pDEW228 in host strain GC4468 (FAlac4169 rpsL) (30). The pcnB mutation was introduced into this strain by FlclrlOO


170 mediated transduction (31) to tetracycline resistance using phage lysates of E. coli strain M R i 9 3 (pcnB80 zad'2084::Tnl0 A(argF4ac)\69 flhD5301 fruA25 relAl, rpsLlSO (strR), Arbs-7, deoCl) (32). The cotransduction o f the pcnB80 allele, yielding strain DPD2227, was detected by screening for reduced bioluminescence (about 20 fold).. E. coli strain DPD2238 was made by transduction o f strain DPD2200 to tetracycline resistance with FlclrlOO phage grown on strain D E I 12 (strR, galK2, lacA74 fo/C::miniTn/0) (33) and a second transduction to kanamycin resistance with FlclrlOO phage grown on strain M M 3 8 K 2 6 (ApcnB::kan argG6 asnA31 or asnB32 his-1 leuB6 metBl pyrE gal-6 lacYl xyl-7 supE44 bglJhuA2 gyrA rpsL104 tsx-1 uhp) (34). The presence of the tolC mutation in strain DPD2238 was scored by sensitivity to the bile salts in MacConkey agar (35); the presence of the pcnB mutation was confirmed by the approximately 20-fold reduced bioluminescence.


f

9

9

9

9

Table I. Stress-responsive E. coli Lux Fusion Strains Promoter Fused to lux

tolC allele

Strain Name

Reference

katG

+

Mar/Sox/ Rob

inaA

+

DPD2227 DPD2238 DPD2226 DPD2240

This work This work This work This work

SOS

recA

+

Heatshock

grpE

+

DPD1710 DPD2222 DPD3084 DPD2234

This work This work This work This work

o513

+

DPD2173 DPD2232

(21) This work

yciG

+

DPD2161 DPD2233

(21) This work

Stress Response

Regulatory Circuit

Oxidative damage Internal acidification etc. DNA damage Protein damage "Superstationary phase" Sigma S stress response

OxyR & 0

S

32

(a ) ?

Stationary phase ( 0 ) S

E. coli strain DPD2226, carrying a plasmid-borne fusion of the E. coli inaA promoter to the P. luminescens luxCDABE in a host strain with a pcnB mutation, was constructed by transduction of strain DPD2146 (pDEW218 in host strain GC4468) (21) to tetracycline resistance with VlclrlOO phage grown on strain M R i 9 3 and screening for cotransduction of the pcnB80 mutation, as described above. E. coli strain DPD2240 was made by transduction of strain DPD2146 to tetracycline resistance with PI clrlOO phage grown on strain D E I 12 followed by transduction to kanamycin resistance with VlclrlOO phage grown on strain M M 3 8 K 2 6 . The tolC and pncB mutations were confirmed as described above.



171 E. coli strains DPD1710 and DPD3084 contain chromosomal insertions at the lacZ locus of the E. coli recA or grpE promoters, respectively, fused to the P. luminescens luxCDABE. These strain were constructed in the host strain M M 2 8 (FgalK2 JN(rrnD-rrnE)l rpsL200) (36,37) by a previously described method (38) using plasmids pDEW14 or pDEW107, respectively. Plasmid pDEW14 was constructed by ligating a Pstl-EcoRl fragment containing a recA-luxCDABE fusion from plasmid p R e c A L x x l into plasmid p B R I N T . C m (39). Previously, plasmid p R e c A L x x l had been made by ligating into BamEl and Sail digested pJT205 (29) a fragment containing the recA promoter region that had been obtaining by P G R amplification using pRecALux3 (40) as the template. Plasmid pDEW107 was constructed by ligating the 7.2 kbp Pstl-EcoRI fragment containing a grpE-luxCDABE fusion from plasmid pDEW106 into plasmid p B R I N T . K m (39). Prior to that, plasmid pDEW106 had been made by ligating a 0.6 kbp BamHl fragment containing the grpE promoter region from plasmid pGrpELuxS (33) into plasmid pJT205 (29). E. coli strains DPD2222 and DPD2234 were derived from DPD1710 and DPD3084, respectively, by VlclrlOO transduction using phage grown on strain D E I 12 as the donor,selection for tetracycline resistance, and confirmation of the tolC mutation. E. coli strains DPD2173 and DPD2161 contain plasmids p D E W 2 2 3 and pDEW215 in E. coli host strain GC4468 (21). E. coli strains DPD2232 and DPD2233 were derived from these by FlclrlOO transduction using phage grown on strain D E I 12 as the donor, selection for tetracycline resistance, and screening for lack o f growth on MacConkey agar plates.

Media and Growth of Bacterial Cultures L B medium (31) was used for all stress response experiments. Overnight cultures were grown in L B medium containing 100 Hg/ml of ampicillin for the plasmid-containing strains, or L B medium for the non-plasmid containing strains. These cultures were diluted the following day into L B medium without ampicillin that had been prewarmed to 37°C. A 1:200 dilution was made for the plasmid-containing strains, while a 1:250 dilution was made for the non-plasmid containing strains. Following incubation with shaking at 37°C for 2 hours and 20 minutes the turbidity of the cultures was measured with a Klett-Summerson colorimeter with a red filter and was typically at or near a reading of 30. These actively growing cells were used immediately for bioluminescence analysis. For analysis of the stress responses induced by aluminum chloride, L B medium adjusted to pH5.2 with hydrochloric acid was used because the solubility and toxicity of aluminum chloride is greater at lower p H (41). The cell cultures were grown in the reduced p H medium and used, as described below, such that the untreated control was the culture at low p H , thereby allowing aluminum-specific stress responses to be detected.

Chemicals A 30% solution of hydrogen peroxide ( E M Science) was diluted into L B medium to the desired concentrations. A stock solution of 20 mg/ml nalidixic acid (Sigma Chemical Co.) i n 1 M N a O H was further diluted into L B medium. Likewise, stock


172 solutions of 1M sodium salicylate ( E M Science) in water, 100 m M cadmium chloride (Johnson Matthey Electronics) in water, and 100 mg/ml methyl viologen (Sigma Chemical Co.) i n water were further diluted into L B medium. 2,4-D (Janssen Chimica) was dissolved directly i n L B medium. Aluminum chloride (Sigma Chemical Co.) was dissolved directly into L B medium that had previously been adjusted to p H 5.2 with hydrochloric acid. Ethanol (200 proof, Quantum Chemicals) was diluted directly into L B medium. Aflatoxin B l (Sigma Chemical Company) was dissolved in D M S O prior to metabolic activation.


Bioluminescence Analysis Sterile white 96-well microplates (Microlite, Dynex) were used. The typical arrangement was that the top row (row A ) contained a control chemical for each stress-responsive strain. DPD2227 and DPD2238 (katG4ux) were tested with 0.004% H 0 . DPD2226 and DPD2240 (inaA4ux) were tested with 5 m M sodium salicylate. DPD1710 (recA-lux in the tolCf host) was tested with 30 ug/ml nalidixic acid; while DPD2222 (recA-lux in the tolC host) was tested with 5 ^ig/ml nalidixic acid. DPD3084 and DPD2234 (grpE-lux) were tested with 3% ethanol. DPD2173 and DPD2232 (o5134ux) and DPD2161 and DPD2233 (yciG4ux) were tested with 10 m M sodium salicylate. In each case, these chemicals resulted in increased bioluminescence of the indicated biosensor strains, except for the strains containing the o5134ux fusion where sodium salicylate was an inhibitor of bioluminescence. The microplates were prepared shortly before use. Two-fold or three-fold dilutions of the chemical to be tested were made in rows B through G ; row H was reserved as the untreated control. When the cell cultures were ready, they were added to all wells of duplicate rows in the prepared microplates, covered with a thin clear plastic film (Fasson FasRoll®), and placed in a ML3000 luminometer (Dynex), the incubation chamber of which had been prewarmed to 37°C. Luminescence data was collected at 37°C in the cycle mode using the following settings: High gain; 20 cycles; 300 second pause; 20 A / D reads per well; all data; auto gain- O N ; mixingO N . The data were processed using a specially written Excel macro to convert to a form for graphing and the average of the duplicate data was plotted as relative light units (RLU) versus time (42). These plots were examined to insure that the control chemicals yielded the expected response. Response ratios, which are the ratio of the R L U of the chemically-treated cells culture divided by the R L U of the untreated culture (42), were calculated at the point of maximal positive response, which was at 40 to 45 minutes for each stress-responsive strain, except for the recA4ux fusion strain where the final time point of 110 minutes gave the maximal response. The other exception was the o5134ux fusion strain for which the response ratios were calculated at the point of maximal decrease o f bioluminescence, which was at 110 minutes. The concentration of chemical that gave the largest overall stress induction ratios was chosen for display of the stress fingerprints. 2

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Metabolic Activation with Rat Liver Post-Mitochondria! S9 Extract Postmitochondrial supernatant (S-9) of Sprague Dawley rat liver induced with Aroclor 1254 was purchased from Molecular Toxicology, Inc., stored at -80°C, and thawed on ice prior to use. A cofactor solution of 78 mM sodium phosphate, pH 7.2, 117 mM NaCl, 7.8 mM glucose-6-phosphate, monosodium salt, 5.9 mM P-NADP, sodium salt, 11.4 mM MgCk, 47 mM KC1, was made by minor modification of a previous method (43), filter sterilized, stored at -80°C, and thawed on ice prior to use. A 30% solution of the S-9 extract in cofactor mixture was made by mixing 330 u,l of thawed S-9 with 770 |xl of the thawed cofactor mix. This remained on ice until used. The chemical to be activated was dissolved in DMSO and 10 ul (or less) was added to 90 [ll (or more, to total of 100 jil) of the 30% S-9 mixture. Three controls were also set up: No S-9 extract, which was the chemical in DMSO added to sodium phosphate, NaCl buffer; no chemical, which was DMSO added to the 30% S-9 mixture, and no S-9 and no chemical, which was DMSO added to sodium phosphate, NaCl buffer. These were incubated at 37°C for 10 minutes. The activated mixture or controls were diluted directly into a luminometer microplate by adding 10 ^1 to 90 jol LB medium. Further dilutions were subsequently made in the luminometer microplate. Actively growing cells were added immediately following the dilutions and luminescence was analyzed as above.

Results and Discussion

Characteristic Fingerprints Induced by Five Chemical Stresses The ability of the stress-responsive lux fusion strains to distinguish between various types of chemical stresses was tested by using five chemicals with welldefined modes of action: hydrogen peroxide, nalidixic acid, ethanol, sodium salicylate, and methyl viologen (also known as paraquat). Similar results were obtained with the set of six tolC* biosensor strains and the set of six tolC biosensor strains, although the concentration of chemical required to induce the maximum stress responses was, in some cases, higher for the tolCT strains. The resultsfromthe tolC strains (DPD2238, DPD2240, DPD2222, DPD2234, DPD2232, and DPD2233) are shown in Figure 1; these "stress fingerprints" are the response ratios of the set of biosensors to each chemical. In these bar graphs and those that follow, a line is drawn at a response ratio of 1.0; response ratios of this value indicate that the treatment had no effect on the strain. Response ratios greater than 1.0 ("lights-on") indicate increased expression of the lux gene fusion, and therefore induction of the particular stress response. Ratios less than 1.0 indicate bioluminescence loss caused by the chemical treatment. This non-specific "lumitoxicity" or "lights-off * can be assumed to be due to inhibition of cellular metabolism required for production of energy or reducing power (44).



174

5 Mg/ml Nalidixic Acid

0.004%H2O2 yciG-lux o513-lux\ grpE-lux recA-lux inaA-lux \ katG-lux | 2

yciG-lux 0513-lux grpE-lux recA-lux inaA~lux katG-lux

4 6 Response ratio

10

30 50 Response ratio

10 mM Sodium Salicylate

4% Ethanol yciG-lux 0513-lux grpE-lux recA-lux inaA-lux katG-lux

yciG-lux 0513-lux grpE-lux recA-lux inaA-lux katG-lux 10

20

30

40

50

0

1

Response ratio

2 3 Response ratio

4

50, uo/ml Methyl violoqen yciG-lux o513-lux grpE-lux recA-lux inaA-lux katG-lux 4 6 8 10 Response ratio

12

Figure 1. Stress fingerprints induced by treatment of a panel of six biosensor strains with hydrogen peroxide, nalidixic acid, ethanol, sodium salicylate, and methyl viologen.


5

175 Hydrogen Peroxide Hydrogen peroxide induced increased bioluminescence from the katG-lux fusion strain i n accordance with the known regulation o f katG expression by the O x y R protein and its activation by hydrogen peroxide (45). Additionally, increased bioluminescence was induced i n two other lux fusion strains. The increased expression of the recA-lux fusion, indicated induction of the D N A damage-sensing SOS response. The increased expression of the grpE-lux fusion strain indicated induction of the protein damage sensing heat shock response. These were also in accordance with known effects o f the hydoxyl radical, formed from the reaction of hydrogen peroxide with transition metals such as F e , in damaging D N A and proteins (16), and in accordance with previous observations using V. fischeri luxCDABE fusions to t h e £ . coli recA mdgrpE promoters (6,46,47). Thus, this three pronged fingerprint of hydrogen peroxide was consistent with its biological mode of action of non-specific damage to macromolecules through an oxidative mechanism.


2+

Nalidixic Acid In contrast to the three pronged fingerprint induced by hydrogen peroxide, nalidixic acid treatment primarily induced expression of the recA-lux fusion, indicating that the principal stress was that of D N A damage. Nalidixic acid-mediated inhibition of D N A gyrase results in double stranded breaks in D N A that are processed by the R e c B C D nuclease to expose a region of single stranded D N A , which likely acts as a signal for induction of the SOS response (2). Thus, nalidixic acid is a more specific D N A damaging agent than hydrogen peroxide and this was reflected in the stress fingerprint. Note, however, that there was a modest 2-fold induction of bioluminescence in the grpE-lux fusion strain, which is in agreement with previous results showing induction of the heat shock response by nalidixic acid (47,48). This result was, therefore, consistent with previous observations that the heat shock response is induced by a large variety of insults to the cell (48-50). Thus, this grpE fusion to the P. luminescens luxCDABE responded to a variety of stresses, as has also been observed with a grpE fusion to the Vibrio fischeri luxCDABE operon (6,33,47,51). The bioluminescence o f the remaining four fusion strains, each of which contains a plasmid-borne lux fusion, was not increased by nalidixic acid treatment. These results suggested that the reported increased plasmid copy number upon induction of the SOS response (52) did not result in false positive report of stress induction within the time frame of these assays.

Ethanol Ethanol is known to be a potent inducer of the protein damage sensing heat shock response (33,50). O f the six stress-responsive fusions represented in this panel, only the expression of grpE-lux, which reports on the heat shock response, was induced by ethanol treatment. The absence of ethanol-induced bioluminescence increases from the katG-lux strain was in contrast to results with a fusion of the E. coli katG promoter region to the V. fischeri lux operon (28). This suggests that the effect of ethanol on the V. fischeri lux fusion may be related to a non-specific solvent effect (42,53) such as stabilization of the V. fischeri lux polypeptides, and furthermore suggests increased specificity as an advantage of the P. luminescens lux reporter.


176


Sodium Salicylate The weak acid sodium salicylate causes cytoplasmic acidification by diffusing through the membrane in a protonated form, followed by release of a hydrogen ion inside the cell (18,54). Treatment with this compound induced a different, three pronged stress fingerprint. Expression of two of these fusions, inaA-lux and yciG-lux, had previously been shown to be induced by this treatment, with induction of the inaA-lux expression being under control of mar A and rob (21). Increased expression of the grpE-lux fusion demonstrated, again, the general stress-responsive nature of the heat shock response.

Methyl Viologen A two pronged fingerprint was induced by treatment with the redox cycling agent methyl viologen. Once again, induction of expression of the grpE-lux fusion was observed. Increased bioluminescence from the inaA-lux fusion strain was also seen. In contrast to induction of inaA expression by sodium salicylate, the induction by methyl viologen is under control of the SoxR and SoxS regulon (20). This regulon responds to oxidative damage caused by superoxide anion radical that is generated by one-electron reduction of molecular oxygen mediated by methyl viologen (16). Lack of induction of the katG-lux fusion in response to methyl viologen contrasts to the moderate induction observed using a katG fusion to the V. fischeri luxCDABE reporter (6). Similarly to the case with ethanol, this lack of response suggests that fusions to the P. luminescens luxCDABE reporter confer increased specificity o f responses. Overall, each of these five chemicals with defined modes o f action yielded a characteristic stress response fingerprint that was reflective of the biological effects caused by these compounds. Thus, these results validated the responsiveness of this panel to characteristic insults.

Stress Fingerprint Induced by a Herbicide, 2,4-D, and two toxic metal salts Chemicals with modes of action that are not as well-defined were addressed next. Stress fingerprints induced by a herbicide with a mode of action thought to mimic plant hormones and two toxic metal salts are shown in Figure 2. The four pronged fingerprint induced by 2,4-D treatment indicates that it causes multiple stresses on the bacterial cell. This fingerprint was most like that induced by sodium salicylate because the expression of the yciG, grpE and inaA fusions were induced. Thus, 2,4-D may, like sodium salicylate, be functioning to bring hydrogen ions into the cytoplasm and thereby lowering internal p H . Interestingly, the slight induction o f the katG fusion indicates that the cell may also sense an oxidative stress; the mechanism of this effect is not known. Both CdCU and AICI3 induced expression of the grpE-lux fusion, although to varying degrees. This again indicated that the heat shock response is generally inducible by numerous stresses to the cell. Treatment with cadmium chloride has been shown to induce synthesis of the GrpE protein (48); however, a similar fusion of the grpE promoter to the V. fischeri luxCDABE reporter does not respond to cadmium chloride unless ethanol or other inducers o f the heat shock response are


177


200 ug/ml 2,4-D yciG-lux 0513-1 ux grpE-lux recA-lux inaA-lux katG-lux 4

6

8

10

12

Response ratio

46 |jg/mlCdCl2

74 Mg/mlAICl3 yciG-lux 0513-1 ux grpE-lux recA-lux inaA-lux katG-lux

yciG-lux 0513-lux grpE-lux recA-lux inaA-lux katG-lux 4

6

8

Response ratio

10

12

10

20

30

40

50

Response ratio

Figure 2. Stress fingerprints induced by treatment of a panel of six biosensor strains with 2,4-dichlorophenoxyacetic acid, aluminum chloride and cadmium chloride.



178 simultaneously present (51). Thus, an advantage of the P. luminescens luxCDABE reporter was demonstrated because it can directly report on this stress. Cadmium chloride did not induce expression of the recA-lux fusion. A similar negative results for induction of the SOS response is also observed in E. coli strains containing an SOS responsive promoter fusion to the lacZ reporter and correlates with the negative result in the Ames test for mutagenesis (3). However, results obtained by two dimensional gel electrophoresis suggest that the amount of the RecA protein is transiently increased following a cadmium chloride stress (55). This study and an earlier two dimensional gel study (48) did not identify other members of the SOS regulon as induced by cadmium chloride; thus, expression of the RecA protein may be regulated by posttranscriptional mechanisms following cadmium stress. The remainder of the patterns of gene expression induced by these two metal salts differed. Aluminum chloride induced the yciG-lux fusion, while cadmium chloride did not. On the other hand, cadmium chloride induced the inaA-lux fusion, while aluminum chloride did not. Thus, the biological effects of these two inorganic metal salts were distinguished by their stress fingerprints. It worth noting the induction of some but not all of these fusions indicated that aluminum chloride was not a nonspecific activator of the P. luminescens luxCDABE. This result, which contrasts with observations from transcriptional fusions using V. harveyi luxAB in E. coli (56), points to another advantage of the P. luminescens luxCDABE reporter. It is also noteworthy that the stress pattern induced by cadmium chloride was like that induced by methyl viologen. Although these two compounds are structurally unrelated, they both function as oxidizing agents (16,48); this similar mode of action was reflected in the similar stress fingerprints induced.

Stress Fingerprints Induced by Aflatoxin B l With and Without Metabolic Activation Aflatoxin Bl, a mycotoxin that is a carcinogen in many animal species (57) and a risk factor for human hepatocellular carcinoma (58), requires metabolic activation to the mutagenic epoxide form (59). Because bacteria lack the enzymes for this and other transformations of promutagens to mutagens, many bacterial tests have incorporated an in vitro activation step (6,60-64). Thus, stress fingerprints for this important environmental contaminant were done with and without pretreatment with a rat liver microsomal S9 extract. These fingerprints are shown in Figure 3. The unactivated aflatoxin Bl induced modest stress responses reported by the SOScontrolled recA-lux and the multiply regulated inaA-lux fusions. Interesting, even without activation by a mammalian extract, the DNA damage response was observed. This modest induction of the recA-lux fusion without S9 treatment required the presence of the tolC mutation in the host strain (data not shown). Also interesting was the induction of inaA-lux expression by untreated aflatoxin Bl. One of the regulators controlling the expression of this gene fusion is the multiple antibiotic resistance response (Mar). It may be that the Mar response is induced by mycotoxins. Activation of aflatoxin Bl with the rat liver microsomal extract resulted in an altered stress fingerprint. The response of the DNA damage-responsive recA-lux fusion was increased, as expected for an increased amount of the mutagenic form of aflatoxin Bl, and was the major stress induced. In contrast, the induction of the inaA-lux fusion


179 was decreased, although at lower concentration of activated aflatoxin B l it was still present (data not shown). Use of this panel of six stress responsive strains, thus, provided a measure of the D N A damage response caused by aflaxtoxin B l , as do other bacterial tests (64-67). However, additional information about the biological effects of aflatoxin B l in its activated and unactivated forms was also obtained. Accordingly, the stress fingerprint of the two forms of aflatoxin B l are distinguished from each other (Figure 3), as well as from that of nalidixic acid (Figure 1), a D N A damaging agent with a different mode of action.


Detection Limits for Aflatoxin Bl The limits of aflatoxin B l detection by the recA-lux and inaA-lux fusion strains were determined by using various concentrations o f untreated and activated compound. The bioluminescence of the culture with chemical added was tested to determine i f it was greater than that of the control without chemical. The unpaired t test was used and P O . 0 0 5 values in a one-tailed test were considered significant. With this criterion, Table II shows the lowest concentrations of aflatoxin B l detected.

Table II. Detection Limits for Aflatoxin B l Sensor recA-lux inaA-lux

With Metabolic Activation 11 (Ig/ml (P

A Panel of Bioluminescent Biosensors for Characterization of

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