ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1990, p. 111-116
Vol. 34, No. 1
0066-4804/90/010111-06$02.00/0 Copyright X 1990, American Society for Microbiology
Sensitive Biological Detection Method for Tetracyclines Using a tetA-lacZ Fusion System IAN CHOPRA,1t* KEITH HACKER,1 ZIVA MISULOVIN,2 AND DAVID M. ROTHSTEIN3 Department of Microbiology, School of Medical Sciences, University of Bristol, Bristol, BS8 I TD, United Kingdom,' and Department of Molecular Biology2 and Department of Microbial Biochemistry and Genetics,3 Medical Research Division, Lederle Laboratories, Pearl River, New York 10965 Received 5 June 1989/Accepted 20 October 1989
A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing cloned tetA-lacZ gene fusion, is described. Expression of ,l-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of I-galactosidase synthesis. Because synthesis of I8-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed. a
The tetracyclines have been extensively used for the
pounds other than tetracyclines are not known to cause derepression of tetA (6). Assaying for tetracyclines is simple and convenient because P-galactosidase is readily detected by using chromogenic substrates. Both a liquid assay and a plate assay are described.
treatment of infections in humans and animals and as growth promoters in animal feeds (8, 10, 17). The widespread use of these antibiotics has led to the development of methods to
quantify them in pharmaceutical preparations, biological materials, and animal feed premixes. Microbiological procedures are the most widely used (19, 22, 25; The Medicines [Animal Feeding Stuffs], Enforcement Regulations, Her Majesty's Stationery Office, London, 1985) and officially accepted (25; The Medicines, Regulations, 1985) methods for detection of tetracyclines and are based on the antimicrobial activities of these drugs. Although microbiological methods are often adequate, they have certain limitations. For example, they are not particularly sensitive (detection limit, about 50 ng of tetracyclines per ml), are subject to interference from other antibiotics, are relatively slow, and are not ideally suited to quantitation of tetracyclines in the sample (19, 32). Alternative, nonmicrobiological procedures for the detection of tetracyclines include spectrophotometric methods (30), fluorimetry (28, 32), high-performance liquid chromatography (19, 34), and polarographic analysis (5). These procedures also have certain drawbacks. Spectrophotometric methods and fluorimetry are insensitive and interference from other materials cannot always be excluded (28, 32), and high-performance liquid chromatography and polarographic analysis require special equipment. In view of these difficulties, there is a need to develop additional assay methods for tetracyclines. In this report, we present a new microbiological method which is based on the induction of the pSC101 tetracycline resistance gene (tetA) by tetracyclines, which inactivate the repressor encoded by the tetR gene. Because direct detection and quantification of the TetA protein is complex (6, 7), we have used a plasmid system in which an in vitro gene fusion has been used to join an enzymatically active ,-galactosidase segment to the amino-terminal fragment of the pSC101-encoded TetA protein. The fusion strain is specific for tetracyclines, since com-
MATERIALS AND METHODS
Chemicals and enzymes. Tetracycline, oxytetracycline, and chlortetracycline were purchased from Sigma Chemical Co., St. Louis, Mo., and doxycycline was donated by Pfizer Research, Sandwich, England. Other tetracyclines were prepared in Lederle Laboratories. Nitrocefin was donated by Glaxo Group Research, Greenford, England. [methyl3H]thymidine (90 Ci/mmol) was purchased from Amersham International, Buckinghamshire, England. Restriction endonucleases and DNA polymerases were purchased from P-L Biochemicals, Inc., Milwaukee, Wis. All other chemicals were purchased from standard commercial sources. Growth media and growth conditions. Nutrient broth (9), nutrient agar (9), and lactose MacConkey indicator medium (29) were used. Growth of bacteria, testing of antibiotic susceptibility on plates, isolation of plasmid DNA, and transformation were performed as previously described (14, 24). Bacterial growth rates were determined as described elsewhere (9), from the gradients of semilogarithmic plots of culture turbidity versus time. Bacterial strains and plasmids. Bacteria and plasmids used in this study are described in Table 1. Plasmid pUB3610 was constructed as follows. The tetR gene was derived from a 2.1-kilobase (kb) fragment of pSC101 after digestion with PvuII and SaII restriction enzymes. The 2.1-kb fragment was ligated into the 3.7-kb fragment of pBR322 which was obtained by digesting at the EcoRI site (made blunt with Klenow fragment enzyme) and the SalI site. The resulting plasmid, containing the tetR and tetA genes, was isolated after transformation into strain DH5 and selection for ampicillin and tetracycline resistance. The plasmid was digested with EcoRV and Sall enzymes, and the lacZ gene (lacking the first eight codons) was ligated after digesting pMC1871 (29) with SmaI and SalI enzymes. Transformants containing plasmid pUB3610 (see Fig. 1) were selected on lactose-
* Corresponding author. t Present address: Department of Microbial Biochemistry and Genetics, Medical Research Division, Lederle Laboratories, Pearl River, NY 10965.
111
112
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CHOPRA ET AL.
EcoRl Hindlil
TABLE 1. E. coli K-12 strains and plasmids Strain or
.
plasmid
r
Strains DH5 JC3272 DU1200
Source or reference
A(argF-lacZYA) endAl gyrA96 hsdRl7(rk- mk') recAl relAl sup44 thi-J
18
his AlacX74 lys rpsL strA trp Derived by transposition of TnJO from pDU300" to the chromosome of JC3272
7, 16 16
tetA tetR Amp' tetA tetA-lacZ fusion vector Ampr tetA-lacZ tetR
1 31 29 This study
Plasmids
pSC1O1 pBR322 pMC1871 pUB3610
a pDU301 is a mutant of plasmid R100-1 which expresses tetracycline resistance constitutively (16).
MacConkey indicator medium containing 25 gug of ampicillin per ml. Determination of plasmid copy number. Plasmid copy numbers were determined as described previously (20). Briefly, DNA labeled with [methyl-3H]thymidine was separated into chromosomal and plasmid fractions by boiling and centrifuging cell extracts. Samples of each fraction were counted to determine copy numbers (20). Enzyme and protein assays. Bacteria were suspended in sodium phosphate buffer (10 mM, pH 7.0) and sonicated at 4°C in an MSE Soniprep 150 ultrasonic disintegrator (amplitude, 6 ,um; four 30-s bursts with 30-s cooling periods) prior to the enzyme and protein assays described below. jLactamase (EC 3.5.2.6) and p-galactosidase (EC 3.2.1.23) were assayed as described elsewhere (12), using, respectively, the chromogenic cephalosporin nitrocefin and 0nitrophenyl-i-D-galactopyranoside. Protein was determined by the method of Bradford (3). Plate test for detection of tetracycines. Strain JC3272 (pUB3610) was grown in nutrient broth containing cyclic AMP (cAMP; 0.5 mM) and ampicillin (50 p,g/ml) to a cell density of 0.5 absorbance units (600 nm). Samples (2.5 ml) of the culture were harvested by centrifugation and resuspended in 0.5 ml of broth. The cells were mixed with 7 ml of agar (1%, wt/vol) and poured onto 30 ml of nutrient agar containing cAMP and ampicillin, within a 140-mm-diameter petri plate. The plates were spotted with tetracycline solutions (10 ,ul) and incubated for 3 h at 37°C. The plates were overlaid with a solution containing 4 mg of 6-bromo-2naphthyl-p-D-galactopyranoside and 26 mg of fast blue RR (Sigma) dissolved in 0.6 ml of dimethyl sulfoxide, to which 11.4 ml of 1% (wt/vol) soft agar was added. P-Galactosidase was detected by the presence of a purple spot (15). RESULTS Characterization of plasmid pUB3610. The cloning strategy described in Materials and Methods was designed to produce a recombinant plasmid (pUB3610; Fig. 1) in which expression of j3-galactosidase is controlled by the pSC101 tetR gene by virtue of a tetA-lacZ translational fusion. Electrophoretic analysis of restriction fragments generated by EcoRI, BamHI, or PstI gave a molecular size estimate of 8.44 ± 0.24 kb, as predicted from data on the sequence of DNA constituting plasmid pUB3610 (1, 4, 21, 23, 27, 29, 31, 33). The hybrid ,B-galactosidase encoded by pUB3610 was predicted
Nrul
FIG. 1. Physical map of pUB3610 (8.5 kb). The inner circle shows the boundaries of the DNA derived from pSC101, pBR322, and pMC1871. The sites for various restriction enzymes are shown at the perimeter, with the EcoRI site at the top corresponding to nucleotide position 0 in the numbering system of Sutcliffe (31) and Bernardi and Bernardi (1). The position and direction of transcription of genes encoding the tetracycline repressor (tetR), a hybrid ,-galactosidase (tetA-lacZ), and 13-lactamase (bla) are indicated, together with the origin of replication (or,). The map has been drawn on the basis of the cloning strategy described in Materials and Methods and from published information on the locations of the genes and their sequences within the DNA fragments constituting pUB3610 (1, 4, 21, 23, 27, 29, 31, 33).
to be a soluble protein containing the first 34 amino acids of the TetA protein at its amino-terminal end. In strains harboring pUB3610, p-galactosidase activity was located in the
soluble cell fraction, with negligible activity associated with membrane fractions (data not shown). Evidence that expression of lacZ was controlled by the tetR product was also obtained. ,B-Galactosidase synthesis was induced by tetracycline, and in strain JC3272(pUB3610) grown in nutrient broth, maximum enzyme induction occurred when the strain was cultured in the presence of 30 ng of tetracycline per ml (Fig. 2). In contrast, induction of comparable levels of j-galactosidase in strain DU1200 (pUB3610) required 2 ,ug of tetracycline per ml of growth medium (data not shown). Since in strain DU1200(pUB3610) the TnJO-encoded tetracycline efflux (TetA) protein is able to remove tetracycline from the cell (6), higher external tetracycline concentrations are required to achieve inactivation of the pUB361O-encoded TetR protein within this strain. Thus, the data are consistent with regulation of lacZ by the TetR protein and also with results reported previously for a tetB-lacZ fusion strain (2). Induction of (3-galactosidase by tetracycline versus bacteriostatic activity of the antibiotic. Tetracycline (5 to 300 ng/ml) was added to cultures of JC3272(pUB3610) (approximately 3 x 108 bacteria per ml) growing at 37°C in nutrient broth. After 1.5 h, bacteria were harvested and their 3-galactosidase activities were determined (Fig. 2). The growth rates of organisms cultured in the presence of tetracycline (5 to 300
DETECTION OF TETRACYCLINES
VOL. 34, 1990 1
16=
0.75
12 0
113
0
a)
Ca)
n
r-
co
0.5
8 80en
3:
0
0
0
6
0.25
4
Xm
oIm 0
0C
0 0
5
10
20
30
40
50
150
100
200
250
300
Tetracycline (ng/ml)
FIG. 2. Effect of tetracycline on growth (U) and -galactosidase production (0) in strain JC3272(pUB3610). A culture of JC3272(pUB3610) (about 3 x 108 cells per ml) growing exponentially in nutrient broth was divided into 12 aliquots. Tetracycline (5 to 300 ng/ml) was added to 11 of these aliquots, with the 12th serving as an antibiotic-free control. Incubation at 37°C was continued for a further 2 h, during which the growth rates of the individual cultures were determined as described in Materials and Methods. The growth rate of the antibiotic-free culture was arbitrarily assigned a value of 1, and other growth rates were related to this value. After 2 h, samples from each culture were processed as described in Materials and Methods to determine the specific activity of ,B-galactosidase (nanomoles of substrate degraded per minute per microgram of protein). Values are the means of replicate determinations for each culture. Standard deviations of the mean were no more than 5% of the values shown.
ng/ml) were also determined (Fig. 2). The addition of tetracycline to the growth medium led to the induction of ,B-galactosidase, with the highest activity expressed in the presence of 30 ng of antibiotic per ml (Fig. 2). By means of ,B-galactosidase induction, the presence of tetracycline in the growth medium could be detected at concentrations below those able to inhibit bacterial growth in liquid culture and on petri plates. A drop in the growth rate was just detectable in the presence of 50 ng of tetracycline per ml (Fig. 2), and on petri plates tetracycline had a MIC of 2 ,ug/ml. The addition of more than 30 ng of tetracycline per ml in the growth medium led to a progressive fall in P-galactosidase activity, accompanied by a decline in bacterial growth rates (Fig. 2). Control experiments established that tetracycline (up to 100 ,ug/ml) had no effect on the enzymatic activity of P-galactosidase in vitro (data not shown). Effects of cAMP and carbon source on expression of ,3galactosidase by JC3272(pUB3610). The expression of the hybrid TetA-p-galactosidase protein could be enhanced by the addition of cAMP (0.5 mM) to cultures of strain JC3272(pUB3610) growing in nutrient broth. The addition of cAMP led to somewhat higher ,-galactosidase activities following induction by 5 to 300 ng of tetracycline per ml (Fig. 3). The presence of cAMP caused a maximum twofold stimulation of 3-galactosidase synthesis (Fig. 3). These results suggest that expression of the tetA-lacZ fusion product in pUB3610 is influenced by the level of cAMP in the cell, i.e., that expression may be subject to catabolite repression. Further experiments to clarify this point were conducted (Table 2). Bacteria were cultured for several generations in nutrient broth or in nutrient broth supplemented with various combinations of glucose and cAMP. Synthesis of 3-galactosidase was then induced in the various cultures by the addition of tetracycline (30 ng/ml). The presence of glucose led to repression of ,B-galactosidase synthesis, but this result was reversed by the concomitant addition of cAMP (Table 2). Use of the tet4-lacZ system for quantitative and qualitative detection of tetracyclines. The possibility of using strain JC3272(pUB3610) grown in nutrient broth supplemented with cAMP in a quantitative assay for the presence of tetracycline in the medium was addressed. Within the range
of 1 to 12.5 ng of tetracycline per ml, a linear dose-response curve was obtained whereby increasing tetracycline concentrations led to a proportional increase in P-galactosidase activity (Fig. 4). Linear dose-response curves were also obtained with other tetracyclines, although the range within which a linear relationship was observed varied among the antibiotics (data not shown). Outside the limits mentioned above, the relationship between P-galactosidase synthesis and tetracycline concentration was not linear. However, the use of strain JC3272 (pUB3610) permitted detection of some tetracycline derivatives, such as doxycycline, in concentrations as low as 0.1 ng/ml (Table 3). However, the presence of cAMP in the medium was necessary for the detection of several tetracyclines at the level of 0.1 ng/ml (data not shown). 20 0
0 15 n
0
*z0 10 0 0 0
0
0 0
5
10
20
FFF
30 40 50 100 150 200 250 300 Tetracycline (ng/ml)
FIG. 3. Effect of cAMP on expression of tetracycline-inducible j-galactosidase synthesis in strain JC3272(pUB3610). Cultures of
JC3272(pUB3610) that had been grown for several generations in nutrient broth (0) or nutrient broth supplemented with cAMP (0.5 mM) ( 1) were divided into aliquots and exposed to tetracycline (5 to 300 ng/ml), as described in the legend to Fig. 2. After 2 h, the p-galactosidase activities in the cultures containing tetracycline were determined, as well as the enzyme activities in antibiotic-free (control) cultures. P-Galactosidase activity is expressed as nanomoles of substrate degraded per minute per microgram of protein. Values are the means of replicate determinations for each culture. Standard deviations of the mean were no more than 5% of the values shown.
ANTIMICROB. AGENTS CHEMOTHER.
CHOPRA ET AL.
114
TABLE 2. Effects of glucose (0.4%, wt/vol) and cAMP (0.5 mM) on expression of ,-galactosidase by JC3272(pUB3610) 1-Galactosidase activity Addition(s) to (nmol/min per
nutrient broth
None.~~~~~~~~~~~~~~~~~~~~~~~~~1 None ...............................................
Tcb (30 ng/ml) ................................... Tc + glucose.................................... Tc, glucose, and cAMP ...................... Tc + cAMP......................................
u.g of protein)'
0.59 ± 0.03 23.13 ± 0.85 15.99 ± 0.76 22.21 ± 0.34 25.07 ± 0.61
Antibiotic
aValues are the means of at least two separate determinations for each culture ±1 standard deviation. b Tc, Tetracycline.
Plate test for detection of tetracyclines. A plate assay system utilizing the fusion strain JC3272(pUB3610) was also developed. After cells were inoculated in nutrient agar containing cAMP, solutions containing tetracyclines were spotted onto the agar surface. After a 3-h incubation, tetracyclines were detected by overlaying with reagents, as described in Materials and Methods. Spots containing 300 ng of tetracycline or chlortetracycline could easily be detected and resulted in a prominent growth-inhibitory zone, at the outside of which was a purple ring indicating induction of 3-galactosidase (Fig. 5). The zones of inhibition were smaller when less drug was applied (e.g., 100 and 30 ng), but tetracyclines were still detectable as faint purple spots even when subinhibitory levels as low as 1 ng were applied (Fig. 5). DISCUSSION Previous studies have established that induction of ,Bgalactosidase synthesis in certain Escherichia coli strains 15
10
0 5
1
2.5
5 7.5 Tetracycline (ng/mi)
TABLE 3. Expression of P-galactosidase by JC3272(pUB3610) in cAMP-supplemented nutrient broth following incubation with various tetracyclines
10
Tetracycline Chlortetracycline Oxytetracycline Minocycline Demethylchlortetracycline (Declomycin) Doxycycline
,B-Galactosidase activity' after incubation with antibiotic at concn (ng/ml) of: 0.1
1
10
0.06 0.53 0.37 0.14 0.02b 1.13
1.84 5.56 3.23 3.32 1.57 5.41
11.62 14.93 13.11 12.46 9.01 18.92
a Values (nanomoles per minute per microgram of protein after subtraction of drug-free, basal activity) are the means of at least two separate determinations for each culture. b Not significantly different from the drug-free, basal activity when analyzed by Student's t test.
carrying tetA-lacZ fusions can occur in the presence of tetracycline concentrations as low as 0.1 ng/ml (2) but did not specifically address the question of whether tetA-lacZ gene fusions can form the basis of a microbiological assay for tetracyclines. In this article, we have demonstrated that a tetA-lacZ fusion within the tetracycline resistance determinant of pSC101 can form the basis of a sensitive quantitative assay for tetracyclines. The system can be used to provide a quantitative test for the presence of as little as 1 ng of tetracycline and a qualitative test for the presence of several tetracyclines in concentrations as low as 0.1 ng/ml. The enhanced susceptibility of strain JC3272(pUB3610) to some tetracycline derivatives (e.g., doxycycline) could be attributable to differences in uptake or interaction with the repressor. These new procedures are more sensitive than existing microbiological assays for detection of tetracyclines and are highly specific for tetracyclines, since the induction of ,Bgalactosidase in the tetA-lacZ fusion system used here depends on the recognition of tetracyclines by the TetR protein. Indeed, apart from cAMP, we are unaware of compounds other than tetracyclines that influence synthesis of the pUB3610-encoded 3-galactosidase. As an alternative to the liquid assay, we developed a plate assay that is convenient for the concurrent testing of many samples. The sensitivity of the plate assay is comparable to that of the liquid assay. However, although the plate assay is simple and reliable, the liquid assay is more quantitative.
12.5
FIG. 4. Relationship between concentration of tetracycline used to induce ,-galactosidase synthesis in JC3272(pUB3610) and the specific activity of enzyme produced. A culture of strain JC3272 (pUB3610) (about 3 x 108 cells per ml) growing exponentially in nutrient broth supplemented with cAMP (0.5 mM) was divided into 7 aliquots. Tetracycline (1 to 12.5 ng/ml) was added to 6 of these cultures, with the 7th serving as an antibiotic-free (control) culture. Following further incubation for 2 h at 37°C, the specific activity of ,-galactosidase in each culture was determined as described in Materials and Methods. The values shown (nanomoles of substrate degraded per minute per microgram of protein) represent means of replicate determinations that have been corrected by subtracting the activity in the tetracycline-free culture. The linearity of the graph was checked by a linear least-squares treatment of the data, using a computer. The slope and intercept were obtained with a correlation coefficient of 0.992.
FIG. 5. Plate assay for detection of tetracyclines. Cells of strain JC3272(pUB3610) were inoculated in nutrient agar containing 0.5 mM cAMP and 50 ,ug of ampicillin per ml. Tetracycline (TC) or chlortetracycline (CTC) solutions (10 ,ul) were spotted; the numbers indicate the total quantity of drug (in nanograms) applied. Zones of growth inhibition at higher drug levels (300 to 30 ng) are evident. The dark shadows (originally purple spots) represent induction of 3galactosidase synthesis.
DETECTION OF TETRACYCLINES
VOL. 34, 1990
Expression of ,-galactosidase by the tetA-lacZ fusion plasmid pUB3610 appears to be somewhat subject to catabolite repression, since enzyme synthesis was slightly repressed when bacteria were grown in the presence of glucose, a situation known to cause decreased cAMP production (13, 26). Further evidence for catabolite repression was provided by the reversal of glucose inhibition upon the addition of cAMP. The possibility that P-galactosidase activity encoded by the tetA-lacZ fusion might vary as an indirect consequence of changes in the copy number of plasmid pUB3610 was addressed by measuring the plasmid copy number and by assaying P-lactamase activity. We did not detect differences in these plasmid functions in response to cAMP addition (data not shown). Since the lacZ gene in pUB3610 is not accompanied by other lac operon DNA (29), it is possible that pSC101 DNA contains a region in, or near, the tetA promoter which is able to bind the cAMP receptor protein-cAMP complex. It is interesting that in the DNA region located between nucleotide -10 and the initiation codon for the TetA protein of pSC101 (1), there is a sequence similar to the consensus sequence for the cAMP receptor protein-cAMP-binding site (11). However, at this point it cannot be ruled out that stimulation of P-galactosidase synthesis in strain JC3272 (pUB3610) by cAMP is due to the fact that the gene fusion is on a multicopy plasmid, a situation in which normal gene regulation may not apply. Nevertheless, in practical terms, this situation dictates that maximum sensitivity for detection of tetracyclines by using the pUB3610 multicopy system requires high cAMP levels in the cell. Although this requirement can be fulfilled by the growth of organisms in minimal media containing carbon sources such as glycerol, we recommend the addition of cAMP to nutrient media as a simple means of achieving nonrepressed rates of ,B-galactosidase synthesis. ACKNOWLEDGMENTS We thank Y. Gluzman for the idea of utilizing the inducible system for detecting tetracyclines, M. Osburne for suggestions about the manuscript, and W. Hughes for technical assistance. LITERATURE CITED 1. Bernardi, A., and F. Bernardi. 1984. Complete sequence of pSC101. Nucleic Acids Res. 12:9415-9426. 2. Bertrand, K. P., K. Postle, L. W. Wray, and W. S. Reznikoff. 1984. Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon TnlO tetracycline resistance determinant. J. Bacteriol. 158:910-919. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 4. Buchel, D. E., B. Gronenborn, and B. Muller-Hill. 1980. Sequence of the lactose permease gene. Nature (London) 283: 541-545. 5. Chatten, L. G., R. E. Moskalyk, R. A. Locock, and K.-S. Huang. 1976. Polarographic analysis of tetracyclines. J. Pharm. Sci.
65:1315-1319. 6. Chopra, I. 1985. Mode of action of the tetracyclines and the nature of bacterial resistance to them, p. 317-392. In J. J. Hlavka and J. H. Boothe (ed.), Handbook of experimental pharmacology, vol. 78. Springer-Verlag KG, Berlin. 7. Chopra, I., P. R. Ball, and S. W. Shales. 1983. Methods of studying plasmid-determined resistance to tetracyclines, p. 223244. In A. D. Russell and L. B. Quesnel (ed.), Antibiotics: assessment of antimicrobial activity and resistance. Society for Applied Bacteriology Technical Series no. 18. Academic Press, Inc. (London), Ltd., London. 8. Chopra, I., T. G. B. Howe, A. H. Linton, K. B. Linton, M. H.
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Richmond, and D. C. E. Speller. 1981. The tetracyclines: prospects at the beginning of the 1980s. J. Antimicrob. Chemother. 8:5-21. Chopra, I., S. J. Johnson, and P. M. Bennett. 1986. Inhibition of Providencia stuartii cell envelope enzymes by chlorhexidine. J. Antimicrob. Chemother. 19:743-751. Cunha, B. A. 1985. Clinical uses of the tetracyclines, p. 393-404. In J. J. Hlavka and J. H. Boothe (ed.), Handbook of experimental pharmacology, vol. 78. Springer-Verlag KG, Berlin. de Crombrugghe, B., S. Busby, and H. Buc. 1984. Cyclic AMP receptor protein: role in transcription activation. Science 224: 831-838. Dixon, R. A., and I. Chopra. 1986. Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. Antimicrob. Agents Chemother. 29:781-788. Dobrogosz, W. J. 1981. Enzymatic activity, p. 365-392. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. Eccles, S. J., and I. Chopra. 1984. Biochemical and genetic characterization of the tet determinant of Bacillus plasmid pAB124. J. Bacteriol. 158:134-140. Elespuru, R. K., and R. J. White. 1983. Biochemical prophage induction assay: a rapid test for antitumour agents that interact with DNA. Cancer Res. 43:2819-2830. Foster, T. J. 1977. Isolation and characterization of mutants of R100-1 which express tetracycline resistance constitutively. FEMS Microbiol. Lett. 2:271-274. Gustafson, R. H., and J. S. Kiser. 1985. Nonmedical uses of the tetracyclines. In J. J. Hlavka and J. H. Boothe (ed.), Handbook of experimental pharmacology, vol. 78. Springer-Verlag KG, Berlin. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. Howell, H. R., L. L. Rhodig, and A. D. Sigler. 1984. Liquid chromatographic determination of chlortetracycline in premixes. J. Assoc. Off. Anal. Chem. 67:572-575. Ivanov, I. G., and D. R. Bachvarov. 1987. Determination of plasmid copy number by the "boiling" method. Anal. Biochem. 165:137-141. Kalnins, A., K. Otto, U. Ruther, and B. Muller-Hill. 1983. Sequence of the lacZ gene of Escherichia coli. EMBO J. 2:593-597. Katz, J. M., and S. E. Katz. 1984. Rapid assay for tetracycline in premixes and mixed feeds. J. Assoc. Off. Anal. Chem. 67:576-579. Klock, G., B. Unger, C. Gatz, W. Hillen, J. Altenbuchner, K. Schmid, and R. Schmitt. 1985. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants. J. Bacteriol. 161:326-332. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Noel, R. J. 1984. Drugs in feed, p. 788-829. In Official methods of analysis, 14th ed. Association of Official Analytical Chemists, Arlington, Va. Pastan, I., and R. L. Perlman. 1968. The role of the lac promoter locus in the regulation of ,-galactosidase synthesis by cyclic 3',5'-adenosine monophosphate. Proc. Natl. Acad. Sci. USA
61:1336-1342. 27. Peden, K. W. C. 1983. Revised sequence of the tetracyclineresistance gene of pBR322. Gene 22:277-280. 28. Samra, Z., J. Krausz-Steinmetz, and D. Sompolinsky. 1979. Transport of tetracyclines through the bacterial cell membrane assayed by fluorescence: a study with susceptible and resistant strains of Staphylococcus aureus and Escherichia coli. Microbios 21:7-21. 29. Shapira, S. K., J. Chou, F. V. Richaud, and M. J. Casadaban. 1983. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of P-galactosidase. Gene 25:71-82.
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30. Sultan, S. M. 1986. Spectrophotometric determination of tetracycline with sodium molybdate. Analyst 111:97-99. 31. Sutcliffe, J. G. 1978. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43:77-90. 32. van den Bogert, C., and A. M. Kroon. 1981. Fluorometric determination of tetracyclines in small blood and tissue sam-
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ples. J. Pharm. Sci. 70:186-189. 33. Watson, N. 1988. A new revision of the sequence of plasmid pBR322. Gene 70:399-403. 34. Wolfs, K., E. Roets, J. Hoogmartens, and H. Vanderhaeghe. 1986. Separation of tetracycline and related substances by high-performance liquid chromatography on poly(styrene-divinyl benzene). J. Chromatogr. 358:444 447.
Vol. 34, No. 1
0066-4804/90/010111-06$02.00/0 Copyright X 1990, American Society for Microbiology
Sensitive Biological Detection Method for Tetracyclines Using a tetA-lacZ Fusion System IAN CHOPRA,1t* KEITH HACKER,1 ZIVA MISULOVIN,2 AND DAVID M. ROTHSTEIN3 Department of Microbiology, School of Medical Sciences, University of Bristol, Bristol, BS8 I TD, United Kingdom,' and Department of Molecular Biology2 and Department of Microbial Biochemistry and Genetics,3 Medical Research Division, Lederle Laboratories, Pearl River, New York 10965 Received 5 June 1989/Accepted 20 October 1989
A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing cloned tetA-lacZ gene fusion, is described. Expression of ,l-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of I-galactosidase synthesis. Because synthesis of I8-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed. a
The tetracyclines have been extensively used for the
pounds other than tetracyclines are not known to cause derepression of tetA (6). Assaying for tetracyclines is simple and convenient because P-galactosidase is readily detected by using chromogenic substrates. Both a liquid assay and a plate assay are described.
treatment of infections in humans and animals and as growth promoters in animal feeds (8, 10, 17). The widespread use of these antibiotics has led to the development of methods to
quantify them in pharmaceutical preparations, biological materials, and animal feed premixes. Microbiological procedures are the most widely used (19, 22, 25; The Medicines [Animal Feeding Stuffs], Enforcement Regulations, Her Majesty's Stationery Office, London, 1985) and officially accepted (25; The Medicines, Regulations, 1985) methods for detection of tetracyclines and are based on the antimicrobial activities of these drugs. Although microbiological methods are often adequate, they have certain limitations. For example, they are not particularly sensitive (detection limit, about 50 ng of tetracyclines per ml), are subject to interference from other antibiotics, are relatively slow, and are not ideally suited to quantitation of tetracyclines in the sample (19, 32). Alternative, nonmicrobiological procedures for the detection of tetracyclines include spectrophotometric methods (30), fluorimetry (28, 32), high-performance liquid chromatography (19, 34), and polarographic analysis (5). These procedures also have certain drawbacks. Spectrophotometric methods and fluorimetry are insensitive and interference from other materials cannot always be excluded (28, 32), and high-performance liquid chromatography and polarographic analysis require special equipment. In view of these difficulties, there is a need to develop additional assay methods for tetracyclines. In this report, we present a new microbiological method which is based on the induction of the pSC101 tetracycline resistance gene (tetA) by tetracyclines, which inactivate the repressor encoded by the tetR gene. Because direct detection and quantification of the TetA protein is complex (6, 7), we have used a plasmid system in which an in vitro gene fusion has been used to join an enzymatically active ,-galactosidase segment to the amino-terminal fragment of the pSC101-encoded TetA protein. The fusion strain is specific for tetracyclines, since com-
MATERIALS AND METHODS
Chemicals and enzymes. Tetracycline, oxytetracycline, and chlortetracycline were purchased from Sigma Chemical Co., St. Louis, Mo., and doxycycline was donated by Pfizer Research, Sandwich, England. Other tetracyclines were prepared in Lederle Laboratories. Nitrocefin was donated by Glaxo Group Research, Greenford, England. [methyl3H]thymidine (90 Ci/mmol) was purchased from Amersham International, Buckinghamshire, England. Restriction endonucleases and DNA polymerases were purchased from P-L Biochemicals, Inc., Milwaukee, Wis. All other chemicals were purchased from standard commercial sources. Growth media and growth conditions. Nutrient broth (9), nutrient agar (9), and lactose MacConkey indicator medium (29) were used. Growth of bacteria, testing of antibiotic susceptibility on plates, isolation of plasmid DNA, and transformation were performed as previously described (14, 24). Bacterial growth rates were determined as described elsewhere (9), from the gradients of semilogarithmic plots of culture turbidity versus time. Bacterial strains and plasmids. Bacteria and plasmids used in this study are described in Table 1. Plasmid pUB3610 was constructed as follows. The tetR gene was derived from a 2.1-kilobase (kb) fragment of pSC101 after digestion with PvuII and SaII restriction enzymes. The 2.1-kb fragment was ligated into the 3.7-kb fragment of pBR322 which was obtained by digesting at the EcoRI site (made blunt with Klenow fragment enzyme) and the SalI site. The resulting plasmid, containing the tetR and tetA genes, was isolated after transformation into strain DH5 and selection for ampicillin and tetracycline resistance. The plasmid was digested with EcoRV and Sall enzymes, and the lacZ gene (lacking the first eight codons) was ligated after digesting pMC1871 (29) with SmaI and SalI enzymes. Transformants containing plasmid pUB3610 (see Fig. 1) were selected on lactose-
* Corresponding author. t Present address: Department of Microbial Biochemistry and Genetics, Medical Research Division, Lederle Laboratories, Pearl River, NY 10965.
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EcoRl Hindlil
TABLE 1. E. coli K-12 strains and plasmids Strain or
.
plasmid
r
Strains DH5 JC3272 DU1200
Source or reference
A(argF-lacZYA) endAl gyrA96 hsdRl7(rk- mk') recAl relAl sup44 thi-J
18
his AlacX74 lys rpsL strA trp Derived by transposition of TnJO from pDU300" to the chromosome of JC3272
7, 16 16
tetA tetR Amp' tetA tetA-lacZ fusion vector Ampr tetA-lacZ tetR
1 31 29 This study
Plasmids
pSC1O1 pBR322 pMC1871 pUB3610
a pDU301 is a mutant of plasmid R100-1 which expresses tetracycline resistance constitutively (16).
MacConkey indicator medium containing 25 gug of ampicillin per ml. Determination of plasmid copy number. Plasmid copy numbers were determined as described previously (20). Briefly, DNA labeled with [methyl-3H]thymidine was separated into chromosomal and plasmid fractions by boiling and centrifuging cell extracts. Samples of each fraction were counted to determine copy numbers (20). Enzyme and protein assays. Bacteria were suspended in sodium phosphate buffer (10 mM, pH 7.0) and sonicated at 4°C in an MSE Soniprep 150 ultrasonic disintegrator (amplitude, 6 ,um; four 30-s bursts with 30-s cooling periods) prior to the enzyme and protein assays described below. jLactamase (EC 3.5.2.6) and p-galactosidase (EC 3.2.1.23) were assayed as described elsewhere (12), using, respectively, the chromogenic cephalosporin nitrocefin and 0nitrophenyl-i-D-galactopyranoside. Protein was determined by the method of Bradford (3). Plate test for detection of tetracycines. Strain JC3272 (pUB3610) was grown in nutrient broth containing cyclic AMP (cAMP; 0.5 mM) and ampicillin (50 p,g/ml) to a cell density of 0.5 absorbance units (600 nm). Samples (2.5 ml) of the culture were harvested by centrifugation and resuspended in 0.5 ml of broth. The cells were mixed with 7 ml of agar (1%, wt/vol) and poured onto 30 ml of nutrient agar containing cAMP and ampicillin, within a 140-mm-diameter petri plate. The plates were spotted with tetracycline solutions (10 ,ul) and incubated for 3 h at 37°C. The plates were overlaid with a solution containing 4 mg of 6-bromo-2naphthyl-p-D-galactopyranoside and 26 mg of fast blue RR (Sigma) dissolved in 0.6 ml of dimethyl sulfoxide, to which 11.4 ml of 1% (wt/vol) soft agar was added. P-Galactosidase was detected by the presence of a purple spot (15). RESULTS Characterization of plasmid pUB3610. The cloning strategy described in Materials and Methods was designed to produce a recombinant plasmid (pUB3610; Fig. 1) in which expression of j3-galactosidase is controlled by the pSC101 tetR gene by virtue of a tetA-lacZ translational fusion. Electrophoretic analysis of restriction fragments generated by EcoRI, BamHI, or PstI gave a molecular size estimate of 8.44 ± 0.24 kb, as predicted from data on the sequence of DNA constituting plasmid pUB3610 (1, 4, 21, 23, 27, 29, 31, 33). The hybrid ,B-galactosidase encoded by pUB3610 was predicted
Nrul
FIG. 1. Physical map of pUB3610 (8.5 kb). The inner circle shows the boundaries of the DNA derived from pSC101, pBR322, and pMC1871. The sites for various restriction enzymes are shown at the perimeter, with the EcoRI site at the top corresponding to nucleotide position 0 in the numbering system of Sutcliffe (31) and Bernardi and Bernardi (1). The position and direction of transcription of genes encoding the tetracycline repressor (tetR), a hybrid ,-galactosidase (tetA-lacZ), and 13-lactamase (bla) are indicated, together with the origin of replication (or,). The map has been drawn on the basis of the cloning strategy described in Materials and Methods and from published information on the locations of the genes and their sequences within the DNA fragments constituting pUB3610 (1, 4, 21, 23, 27, 29, 31, 33).
to be a soluble protein containing the first 34 amino acids of the TetA protein at its amino-terminal end. In strains harboring pUB3610, p-galactosidase activity was located in the
soluble cell fraction, with negligible activity associated with membrane fractions (data not shown). Evidence that expression of lacZ was controlled by the tetR product was also obtained. ,B-Galactosidase synthesis was induced by tetracycline, and in strain JC3272(pUB3610) grown in nutrient broth, maximum enzyme induction occurred when the strain was cultured in the presence of 30 ng of tetracycline per ml (Fig. 2). In contrast, induction of comparable levels of j-galactosidase in strain DU1200 (pUB3610) required 2 ,ug of tetracycline per ml of growth medium (data not shown). Since in strain DU1200(pUB3610) the TnJO-encoded tetracycline efflux (TetA) protein is able to remove tetracycline from the cell (6), higher external tetracycline concentrations are required to achieve inactivation of the pUB361O-encoded TetR protein within this strain. Thus, the data are consistent with regulation of lacZ by the TetR protein and also with results reported previously for a tetB-lacZ fusion strain (2). Induction of (3-galactosidase by tetracycline versus bacteriostatic activity of the antibiotic. Tetracycline (5 to 300 ng/ml) was added to cultures of JC3272(pUB3610) (approximately 3 x 108 bacteria per ml) growing at 37°C in nutrient broth. After 1.5 h, bacteria were harvested and their 3-galactosidase activities were determined (Fig. 2). The growth rates of organisms cultured in the presence of tetracycline (5 to 300
DETECTION OF TETRACYCLINES
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16=
0.75
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113
0
a)
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n
r-
co
0.5
8 80en
3:
0
0
0
6
0.25
4
Xm
oIm 0
0C
0 0
5
10
20
30
40
50
150
100
200
250
300
Tetracycline (ng/ml)
FIG. 2. Effect of tetracycline on growth (U) and -galactosidase production (0) in strain JC3272(pUB3610). A culture of JC3272(pUB3610) (about 3 x 108 cells per ml) growing exponentially in nutrient broth was divided into 12 aliquots. Tetracycline (5 to 300 ng/ml) was added to 11 of these aliquots, with the 12th serving as an antibiotic-free control. Incubation at 37°C was continued for a further 2 h, during which the growth rates of the individual cultures were determined as described in Materials and Methods. The growth rate of the antibiotic-free culture was arbitrarily assigned a value of 1, and other growth rates were related to this value. After 2 h, samples from each culture were processed as described in Materials and Methods to determine the specific activity of ,B-galactosidase (nanomoles of substrate degraded per minute per microgram of protein). Values are the means of replicate determinations for each culture. Standard deviations of the mean were no more than 5% of the values shown.
ng/ml) were also determined (Fig. 2). The addition of tetracycline to the growth medium led to the induction of ,B-galactosidase, with the highest activity expressed in the presence of 30 ng of antibiotic per ml (Fig. 2). By means of ,B-galactosidase induction, the presence of tetracycline in the growth medium could be detected at concentrations below those able to inhibit bacterial growth in liquid culture and on petri plates. A drop in the growth rate was just detectable in the presence of 50 ng of tetracycline per ml (Fig. 2), and on petri plates tetracycline had a MIC of 2 ,ug/ml. The addition of more than 30 ng of tetracycline per ml in the growth medium led to a progressive fall in P-galactosidase activity, accompanied by a decline in bacterial growth rates (Fig. 2). Control experiments established that tetracycline (up to 100 ,ug/ml) had no effect on the enzymatic activity of P-galactosidase in vitro (data not shown). Effects of cAMP and carbon source on expression of ,3galactosidase by JC3272(pUB3610). The expression of the hybrid TetA-p-galactosidase protein could be enhanced by the addition of cAMP (0.5 mM) to cultures of strain JC3272(pUB3610) growing in nutrient broth. The addition of cAMP led to somewhat higher ,-galactosidase activities following induction by 5 to 300 ng of tetracycline per ml (Fig. 3). The presence of cAMP caused a maximum twofold stimulation of 3-galactosidase synthesis (Fig. 3). These results suggest that expression of the tetA-lacZ fusion product in pUB3610 is influenced by the level of cAMP in the cell, i.e., that expression may be subject to catabolite repression. Further experiments to clarify this point were conducted (Table 2). Bacteria were cultured for several generations in nutrient broth or in nutrient broth supplemented with various combinations of glucose and cAMP. Synthesis of 3-galactosidase was then induced in the various cultures by the addition of tetracycline (30 ng/ml). The presence of glucose led to repression of ,B-galactosidase synthesis, but this result was reversed by the concomitant addition of cAMP (Table 2). Use of the tet4-lacZ system for quantitative and qualitative detection of tetracyclines. The possibility of using strain JC3272(pUB3610) grown in nutrient broth supplemented with cAMP in a quantitative assay for the presence of tetracycline in the medium was addressed. Within the range
of 1 to 12.5 ng of tetracycline per ml, a linear dose-response curve was obtained whereby increasing tetracycline concentrations led to a proportional increase in P-galactosidase activity (Fig. 4). Linear dose-response curves were also obtained with other tetracyclines, although the range within which a linear relationship was observed varied among the antibiotics (data not shown). Outside the limits mentioned above, the relationship between P-galactosidase synthesis and tetracycline concentration was not linear. However, the use of strain JC3272 (pUB3610) permitted detection of some tetracycline derivatives, such as doxycycline, in concentrations as low as 0.1 ng/ml (Table 3). However, the presence of cAMP in the medium was necessary for the detection of several tetracyclines at the level of 0.1 ng/ml (data not shown). 20 0
0 15 n
0
*z0 10 0 0 0
0
0 0
5
10
20
FFF
30 40 50 100 150 200 250 300 Tetracycline (ng/ml)
FIG. 3. Effect of cAMP on expression of tetracycline-inducible j-galactosidase synthesis in strain JC3272(pUB3610). Cultures of
JC3272(pUB3610) that had been grown for several generations in nutrient broth (0) or nutrient broth supplemented with cAMP (0.5 mM) ( 1) were divided into aliquots and exposed to tetracycline (5 to 300 ng/ml), as described in the legend to Fig. 2. After 2 h, the p-galactosidase activities in the cultures containing tetracycline were determined, as well as the enzyme activities in antibiotic-free (control) cultures. P-Galactosidase activity is expressed as nanomoles of substrate degraded per minute per microgram of protein. Values are the means of replicate determinations for each culture. Standard deviations of the mean were no more than 5% of the values shown.
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CHOPRA ET AL.
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TABLE 2. Effects of glucose (0.4%, wt/vol) and cAMP (0.5 mM) on expression of ,-galactosidase by JC3272(pUB3610) 1-Galactosidase activity Addition(s) to (nmol/min per
nutrient broth
None.~~~~~~~~~~~~~~~~~~~~~~~~~1 None ...............................................
Tcb (30 ng/ml) ................................... Tc + glucose.................................... Tc, glucose, and cAMP ...................... Tc + cAMP......................................
u.g of protein)'
0.59 ± 0.03 23.13 ± 0.85 15.99 ± 0.76 22.21 ± 0.34 25.07 ± 0.61
Antibiotic
aValues are the means of at least two separate determinations for each culture ±1 standard deviation. b Tc, Tetracycline.
Plate test for detection of tetracyclines. A plate assay system utilizing the fusion strain JC3272(pUB3610) was also developed. After cells were inoculated in nutrient agar containing cAMP, solutions containing tetracyclines were spotted onto the agar surface. After a 3-h incubation, tetracyclines were detected by overlaying with reagents, as described in Materials and Methods. Spots containing 300 ng of tetracycline or chlortetracycline could easily be detected and resulted in a prominent growth-inhibitory zone, at the outside of which was a purple ring indicating induction of 3-galactosidase (Fig. 5). The zones of inhibition were smaller when less drug was applied (e.g., 100 and 30 ng), but tetracyclines were still detectable as faint purple spots even when subinhibitory levels as low as 1 ng were applied (Fig. 5). DISCUSSION Previous studies have established that induction of ,Bgalactosidase synthesis in certain Escherichia coli strains 15
10
0 5
1
2.5
5 7.5 Tetracycline (ng/mi)
TABLE 3. Expression of P-galactosidase by JC3272(pUB3610) in cAMP-supplemented nutrient broth following incubation with various tetracyclines
10
Tetracycline Chlortetracycline Oxytetracycline Minocycline Demethylchlortetracycline (Declomycin) Doxycycline
,B-Galactosidase activity' after incubation with antibiotic at concn (ng/ml) of: 0.1
1
10
0.06 0.53 0.37 0.14 0.02b 1.13
1.84 5.56 3.23 3.32 1.57 5.41
11.62 14.93 13.11 12.46 9.01 18.92
a Values (nanomoles per minute per microgram of protein after subtraction of drug-free, basal activity) are the means of at least two separate determinations for each culture. b Not significantly different from the drug-free, basal activity when analyzed by Student's t test.
carrying tetA-lacZ fusions can occur in the presence of tetracycline concentrations as low as 0.1 ng/ml (2) but did not specifically address the question of whether tetA-lacZ gene fusions can form the basis of a microbiological assay for tetracyclines. In this article, we have demonstrated that a tetA-lacZ fusion within the tetracycline resistance determinant of pSC101 can form the basis of a sensitive quantitative assay for tetracyclines. The system can be used to provide a quantitative test for the presence of as little as 1 ng of tetracycline and a qualitative test for the presence of several tetracyclines in concentrations as low as 0.1 ng/ml. The enhanced susceptibility of strain JC3272(pUB3610) to some tetracycline derivatives (e.g., doxycycline) could be attributable to differences in uptake or interaction with the repressor. These new procedures are more sensitive than existing microbiological assays for detection of tetracyclines and are highly specific for tetracyclines, since the induction of ,Bgalactosidase in the tetA-lacZ fusion system used here depends on the recognition of tetracyclines by the TetR protein. Indeed, apart from cAMP, we are unaware of compounds other than tetracyclines that influence synthesis of the pUB3610-encoded 3-galactosidase. As an alternative to the liquid assay, we developed a plate assay that is convenient for the concurrent testing of many samples. The sensitivity of the plate assay is comparable to that of the liquid assay. However, although the plate assay is simple and reliable, the liquid assay is more quantitative.
12.5
FIG. 4. Relationship between concentration of tetracycline used to induce ,-galactosidase synthesis in JC3272(pUB3610) and the specific activity of enzyme produced. A culture of strain JC3272 (pUB3610) (about 3 x 108 cells per ml) growing exponentially in nutrient broth supplemented with cAMP (0.5 mM) was divided into 7 aliquots. Tetracycline (1 to 12.5 ng/ml) was added to 6 of these cultures, with the 7th serving as an antibiotic-free (control) culture. Following further incubation for 2 h at 37°C, the specific activity of ,-galactosidase in each culture was determined as described in Materials and Methods. The values shown (nanomoles of substrate degraded per minute per microgram of protein) represent means of replicate determinations that have been corrected by subtracting the activity in the tetracycline-free culture. The linearity of the graph was checked by a linear least-squares treatment of the data, using a computer. The slope and intercept were obtained with a correlation coefficient of 0.992.
FIG. 5. Plate assay for detection of tetracyclines. Cells of strain JC3272(pUB3610) were inoculated in nutrient agar containing 0.5 mM cAMP and 50 ,ug of ampicillin per ml. Tetracycline (TC) or chlortetracycline (CTC) solutions (10 ,ul) were spotted; the numbers indicate the total quantity of drug (in nanograms) applied. Zones of growth inhibition at higher drug levels (300 to 30 ng) are evident. The dark shadows (originally purple spots) represent induction of 3galactosidase synthesis.
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Expression of ,-galactosidase by the tetA-lacZ fusion plasmid pUB3610 appears to be somewhat subject to catabolite repression, since enzyme synthesis was slightly repressed when bacteria were grown in the presence of glucose, a situation known to cause decreased cAMP production (13, 26). Further evidence for catabolite repression was provided by the reversal of glucose inhibition upon the addition of cAMP. The possibility that P-galactosidase activity encoded by the tetA-lacZ fusion might vary as an indirect consequence of changes in the copy number of plasmid pUB3610 was addressed by measuring the plasmid copy number and by assaying P-lactamase activity. We did not detect differences in these plasmid functions in response to cAMP addition (data not shown). Since the lacZ gene in pUB3610 is not accompanied by other lac operon DNA (29), it is possible that pSC101 DNA contains a region in, or near, the tetA promoter which is able to bind the cAMP receptor protein-cAMP complex. It is interesting that in the DNA region located between nucleotide -10 and the initiation codon for the TetA protein of pSC101 (1), there is a sequence similar to the consensus sequence for the cAMP receptor protein-cAMP-binding site (11). However, at this point it cannot be ruled out that stimulation of P-galactosidase synthesis in strain JC3272 (pUB3610) by cAMP is due to the fact that the gene fusion is on a multicopy plasmid, a situation in which normal gene regulation may not apply. Nevertheless, in practical terms, this situation dictates that maximum sensitivity for detection of tetracyclines by using the pUB3610 multicopy system requires high cAMP levels in the cell. Although this requirement can be fulfilled by the growth of organisms in minimal media containing carbon sources such as glycerol, we recommend the addition of cAMP to nutrient media as a simple means of achieving nonrepressed rates of ,B-galactosidase synthesis. ACKNOWLEDGMENTS We thank Y. Gluzman for the idea of utilizing the inducible system for detecting tetracyclines, M. Osburne for suggestions about the manuscript, and W. Hughes for technical assistance. LITERATURE CITED 1. Bernardi, A., and F. Bernardi. 1984. Complete sequence of pSC101. Nucleic Acids Res. 12:9415-9426. 2. Bertrand, K. P., K. Postle, L. W. Wray, and W. S. Reznikoff. 1984. Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon TnlO tetracycline resistance determinant. J. Bacteriol. 158:910-919. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 4. Buchel, D. E., B. Gronenborn, and B. Muller-Hill. 1980. Sequence of the lactose permease gene. Nature (London) 283: 541-545. 5. Chatten, L. G., R. E. Moskalyk, R. A. Locock, and K.-S. Huang. 1976. Polarographic analysis of tetracyclines. J. Pharm. Sci.
65:1315-1319. 6. Chopra, I. 1985. Mode of action of the tetracyclines and the nature of bacterial resistance to them, p. 317-392. In J. J. Hlavka and J. H. Boothe (ed.), Handbook of experimental pharmacology, vol. 78. Springer-Verlag KG, Berlin. 7. Chopra, I., P. R. Ball, and S. W. Shales. 1983. Methods of studying plasmid-determined resistance to tetracyclines, p. 223244. In A. D. Russell and L. B. Quesnel (ed.), Antibiotics: assessment of antimicrobial activity and resistance. Society for Applied Bacteriology Technical Series no. 18. Academic Press, Inc. (London), Ltd., London. 8. Chopra, I., T. G. B. Howe, A. H. Linton, K. B. Linton, M. H.
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61:1336-1342. 27. Peden, K. W. C. 1983. Revised sequence of the tetracyclineresistance gene of pBR322. Gene 22:277-280. 28. Samra, Z., J. Krausz-Steinmetz, and D. Sompolinsky. 1979. Transport of tetracyclines through the bacterial cell membrane assayed by fluorescence: a study with susceptible and resistant strains of Staphylococcus aureus and Escherichia coli. Microbios 21:7-21. 29. Shapira, S. K., J. Chou, F. V. Richaud, and M. J. Casadaban. 1983. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of P-galactosidase. Gene 25:71-82.
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ples. J. Pharm. Sci. 70:186-189. 33. Watson, N. 1988. A new revision of the sequence of plasmid pBR322. Gene 70:399-403. 34. Wolfs, K., E. Roets, J. Hoogmartens, and H. Vanderhaeghe. 1986. Separation of tetracycline and related substances by high-performance liquid chromatography on poly(styrene-divinyl benzene). J. Chromatogr. 358:444 447.
