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Detection and quantification of tetracyclines by whole cell biosensors
FEMS Microbiology Letters 190 (2000) 273^278
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Detection and quanti¢cation of tetracyclines by whole cell biosensors Lars Hestbjerg Hansen, SÖren Johannes SÖrensen * Department of General Microbiology, University of Copenhagen, SÖlvgade 83 H, DK-1307 Copenhagen K, Denmark Received 22 May 2000 ; received in revised form 12 July 2000; accepted 12 July 2000
Abstract Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing L-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different Gramnegative bacteria by conjugation. An Escherichia coli strain containing the Ptet -lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tetracycline ; Whole-cell biosensor; Reporter gene; lux; lacZ; gfp
1. Introduction In recent years, the use of biosensors to detect the presence of chemical compounds in complex environments has been widespread. Biosensors have enabled the detection and quanti¢cation of various compounds, such as metals [1], xenobiotic compounds [2], and antibiotics [3]. We have focussed on the detection and quanti¢cation of tetracycline by the use of biosensors based on the inducible resistance gene promoter Ptet (from Tn10) in combination with its regulatory gene tetR. This repressor/promoter interaction is well described elsewhere [4] as is its speci¢city in recognizing tetracyclines as inducers [5]. Biosensors based on fusions to this promoter will therefore produce a quantitative response to increasing levels of tetracyclines. Biosensors provide an inexpensive and sensitive alternative to non-microbiological procedures for detecting tetracyclines. These procedures include spectrophotometric methods [6,7] and high-performance liquid chromatography [8,9]. Most biosensor organisms used so far carry the biosensor gene cassettes on plasmids [3,10,11]. Problems with plasmid based systems can be varying copy numbers and loss of sensitivity [12]. It is also known that in some instances plasmids are lost from the organisms, if the organ-
isms are introduced to environments without any selective pressure [2]. The choice of reporter gene can vary, depending on the di¡erent environmental conditions in which the biosensors are used. For example, both gfp and lux will prove to be useless under anaerobic conditions since both folding of the GFP protein and light production from the lux genes require oxygen [13]. The gfp gene might be preferred in long-term incubations under low transcriptional activity, since the GFP is very stable and therefore accumulates within the cell [14]. We therefore developed a variety of £exible biosensor vectors for the detection and quanti¢cation of very low concentrations of tetracyclines in various environments. We present the choice of three di¡erent reporter gene systems: luxCDABE from Vibrio ¢scheri, lacZYA from Escherichia coli and gfp from Aequorea victoria, combined with the tetracycline inducible promoter from Tn10. Furthermore, we also transferred all three combinations into mini-Tn5 delivery vectors, which enable the introduction of the di¡erent biosensor-constructs as single chromosomal inserts into a variety of Gram-negative bacteria. 2. Materials, methods and results 2.1. Strains, plasmids and culture conditions
* Corresponding author. Tel. : +45 3532-2053; Fax: +45 3532-2040; E-mail : [email protected]
The E. coli strain MT102 was used as host strain in all
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 4 7 - 5
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DNA manipulation steps except for hosting the ¢nal biosensor constructs in the mini-Tn5 plasmids pUT-tetlux, -tetlac, -tetgfp. Because these plasmids need the Z protein for replication [24], they were transformed into MT102PIR. The MT102-PIR strains hosting the di¡erent biosensor plasmids were in turn used as donors in tri-parental ¢lter matings to insert the biosensor cassettes into the chromosomes of di¡erent Gram-negative bacteria. Strains of E. coli were grown in LB broth [25] at 37³C, unless otherwise stated. Other strains used were grown at 30³C. Tri-parental ¢lter matings were carried out as described earlier [16], and biosensor transconjugants were selected on basal salts medium [26] containing 0.2% glucose and 50-Wg ml31 kanamycin. All strains and plasmids are listed in Table 1. DNA plasmids and fragments were prepared, transformed, analyzed and manipulated by standard procedures [27]. Tetracycline^HCl was used in all inductions unless otherwise stated. 2.2. Cloning of the tetlux gene cassette The tet repressor/promoter was excised from pNK81 as a 1.6 kb BglII^EcoRI fragment containing tetR, Ptet and the beginning of the tetA gene. This fragment was then cloned into pUCD615, thereby creating an operon fusion between the tet-repressor, regulated promoter Ptet and the luxCDABE genes. This plasmid was called pUCDtetlux. The tetlux cassette could not be excised as one fragment. The lux genes were therefore excised as a SalI^PstI fragment and cloned into pLOW2. The plasmid was called pLOW2-lux. Next a SalI fragment from pUCD-tetlux
containing tetR and Ptet was ligated to SalI-digested pLOW2-lux. The resulting plasmid was named pLOW2tetlux. The entire tetlux construct was transferred from pLOW2-tetlux to pUT-kn-res as a NotI fragment to yield pUT-tetlux (Fig. 1A). 2.3. Cloning of the tetlac gene cassette The same 1.6 kb BglII^EcoRI fragment from pNK81 was ligated to BamHI^EcoRI digested pRS528, thereby creating an operon fusion between Ptet and the lacZYA genes. This plasmid was called pRS-tetlac. The newly created tetlac cassette was then transferred from pRS-tetlac to pLOW2 as a PstI^SalI fragment, yielding pLOW2-tetlac. This was done in order to generate NotI ends on the biosensor cassette. The cassette was then transferred from pLOW2-tetlac into the unique NotI site of the mini-Tn5 delivery vector pUT-kn-res to generate pUT-tetlac (Fig. 1B). 2.4. Cloning of the tetgfp gene cassette As a consequence of the incompatibility of cloning sites between the GFP vector pAG408 and the wild-type tet repressor/promoter fragment from Tn10, new restriction sites were introduced on the tet repressor/promoter fragment. This was done by PCR. The tet repressor/promoter was ampli¢ed from pNK81 using the following primers, Ptet NotI: 5P-ggcgaagcggccgctaagatctatg-3P and Ptet ClaI: 5Pagtggttatcgatatcttccgaagc-3P, to yield a 1126-bp fragment containing tetR, Ptet and the beginning of tetA. Thus, NotI
Table 1 Plasmids and strains Plasmids/strains Plasmids pAG408 pAG-tetgfp pLOW2 pLOW2-lux pLOW2-tetlac pLOW2-tetlux pNK81 pRB28 pRS528 pRS-tetlac pUCD615 pUCD-tetlux pUT-Kn-res pUT-tetgfp pUT-tetlac pUT-tetlux RK600 Strains MC4100 MC4100: :tetlac MT102 MT102-PIR NF1815
Replicon/species
Characteristics
Reference
R6K R6K p15A p15A p15A p15A pMB1 pMB1 pMB1 pMB1 pMB1 pMB1 R6K R6K R6K R6K pMB1
KmR , gfp KmR , Ptet -gfp KmR KmR , luxCDABE KmR , Ptet -lacZYA KmR , Ptet -luxCDABE AmpR , tetR-Ptet -tetA KmR , AmpR , luxCDABE AmpR , lacZYA AmpR , Ptet -lacZYA KmR KmR , Ptet -luxCDABE KmR , AmpR , mini-Tn5 pUT-Kn-res-Ptet -gfp pUT-Kn-res-Ptet -lacZYA pUT-Kn-res-Ptet -luxCDABE CmR
[15] This study [16] This study This study This study ATCC 77337 [17] [18] This study [19] This study [20] This study This study This study [21]
E. E. E. E. E.
glu+SmR MC4100: :Ptet -lacZYA leu pro thi SmR MT102: :pir-aphA p+ leu thi SmR
[22] This study [23] [16] N. Fiil
coli coli coli coli coli
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Fig. 1. Construction of the three biosensor cassettes and their insertion into pUT-Kn-res. (A) The tetlux cassette, an operon fusion between tetR-Ptet from Tn10 and the luxCDABE operon from V. ¢scherii, (B) the Ptet fusion to the E. coli lacZYA operon and (C) the fusion of Ptet to the gfp gene from A. victoria. Vertical striped bars represent the tetR gene, the cross-hatched bar represent part of cloning vector pRS528 and gray bars represent the truncated tetA gene from Tn10. Arrows indicate transcription start and restriction sites are indicated above the cassettes.
and ClaI sites (underlined) were generated at the ends of the tet repressor/promoter fragment. After a double digest using NotI and ClaI, the PCR fragment was ligated into pAG408, placing Ptet in front of the gfp gene. This construct was called pAG-tetgfp. The tetgfp cassette was transferred to pUT-kn-res by performing a second PCR. We used Ptet NotI as the forward primer and gfprevNotI: 5P-atggcggccgcattcattatttgt-3P as the reverse primer to generate a tetgfp fragment £anked by NotI sites (undelined). This construct was then ligated to the unique NotI site of pUT-kn-res to generate pUT-tetgfp (Fig. 1C). 2.5. Induction assays From an overnight LB culture of E. coli MT102-PIR/ pUT-tetlux, 1 ml was added to 40 ml of ABB1 medium [28] containing leucine and proline. 3-ml aliquots were
then transferred to disposable plastic tubes (for luminometer counting). Tetracycline was added to obtain various total concentrations as shown in Fig. 2A. The tubes were mixed by gentle inversion and incubated at room temperature without shaking. At various times, the emission of light per 30 s was measured in a BG-P Portable luminometer (MGM instruments, Hamden USA). Relative light units (RLU) at 50 min after incubation were then plotted against tetracycline concentration (Fig. 2A). An overnight culture of E. coli MT102-PIR/pUT-tetlac was diluted 100-fold in fresh LB medium containing 100 Wg ml31 ampicillin and various concentrations of tetracycline. The biosensor strains were grown for 3 h at 37³C with shaking. 0.5 ml was transferred to 1.5-ml Eppendorf tubes and placed on ice. 10 Wl of toluene was added to each tube and vortexed for 10 s. A L-galactosidase assay was then performed according to Miller [25]. The level of
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Fig. 2. The quantitative response of the three biosensor to increasing tetracycline concentrations. (A) Production of light from MT102-PIR/pUT-tetlux cells. (B) L-Galactosidase activity of MT102-PIR/pUT-tetlac cells. (C) Increasing £uorescence of MT102-PIR/pUT-tetgfp cells.
L-galactosidase was then plotted against tetracycline concentration (Fig. 2B). Finally, an overnight LB culture of E. coli MT102-PIR/ pUT-tetgfp was diluted 100-fold in fresh LB medium containing 100 Wg ml31 ampicillin and various concentrations of tetracycline, and grown overnight at 30³C for 16 h. A 3ml sample was washed twice and re-suspended in 0.9% NaCl (to minimize the background £uorescence of the LB medium). The 3-ml sample was then transferred to a cuvette and £uorescence was measured in a Luminescence Spectrometer LS 50B (Perkin Elmer, Buckinghamshire, UK). Excitation wavelength was 395 nm, emission was measured at 509 nm and relative £uorescent units were plotted against the concentration of tetracycline (Fig. 2C). 2.6. Use of an E. coli MC4100: :tetlac biosensor to measure bioavailable tetracycline in milk The pUT-tetlac construct was used to insert the tetlac biosensor cassette into the chromosome of E. coli MC4100 by performing a tri-parental ¢ltermating [16]. This strain was used to demonstrate the utility of these tetracycline biosensors. E. coli MC4100 with the tetlac cassette was used to determine the bioavailable tetracycline content in milk. Milk was taken from a cow treated with oxytetracycline. The treatment (5 mg kg31 ) was given intravenously at day 0, and samples of milk were taken twice daily (Fig. 3). Milk samples were centrifuged for 20 min and ¢ltered through a 0.2-Wm ¢lter. Di¡erent dilutions of milk-¢ltrate were added to test tubes containing 5 ml of LB with kanamycin (50 Wg ml31 ). To obtain a standard curve, known concentrations of oxytetracycline were also added to different tubes. The tubes were then inoculated with 50 Wl of E. coli MC4100: :tetlac taken from an overnight LB broth culture. The biosensor cells were grown for 3 h, toluenized and analyzed. Tetracycline concentrations in the milk samples were calculated by comparing the L-galactosidase ac-
tivity in milk samples with activities on the standard curve. Results were then plotted as oxytetracycline concentrations vs. sampling time (Fig. 3). 3. Discussion Unlike conventional methods for determination of concentrations of tetracyclines, the biosensors determine only the bio-available fraction of these compounds, that is, the actual concentration that the microbial community encounters in the environment. This method thereby does not measure the proportion of tetracyclines that is tightly bound to matrix material like soil particles or is in other ways biologically inactivated. This makes biosensors the tool of choice for investigating di¡erent substances, their concentration and their in£uence on and interaction with microbial communities. Furthermore, biosensors have the potential to become a cost-e¤cient and very sensitive alternative to conventional methods such as HPLC in determining concentrations of di¡erent compounds. It was clearly shown in this study that all three biosen-
Fig. 3. Oxytetracycline content in milk from a cow treated with 5 mg kg31 oxytetracycline. Tetracycline concentrations were measured using biosensor strain E. coli MC4100: :tetlac.
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sor constructs responded in a quantitative manner to different concentrations of tetracycline. We have constructed tetracycline biosensors containing a choice of reporter genes. Comparisons between the sensitivity of the di¡erent biosensors are impeded by the di¡erent assay growth conditions in this study. The need for separate assay conditions illustrates the di¡erences of the three reporters. We were unable to produce satisfactory induction of the lux genes during rapid growth. The amount of GFP present in our biosensors was insu¤cient with short incubations. Finally, both GFP and luciferase (from luxCDABE) failed to work well beyond 30³C. Consequently the assay conditions had to be adjusted for each reporter gene. The luxCDABE and lacZ genes are renowned for their sensitivity in biosensors [1,29,30], and the gfp is the reporter gene of choice for in situ microbial ecology [31,32]. Adding to the versatility of these biosensors is the fact that they can be conjugated into a variety of Gram-negative bacteria, where integration into the host chromosome will ensure a stable construct copy number and maintenance, even under non-selective conditions. We performed successful conjugations of the three biosensor mini-Tn5s to six di¡erent Gram-negative bacteria (data not shown). Until now, biosensor-constructs have been applied in a limited number of bacteria, such as E. coli [33], P. £uorescens [34] and P. putida [35]. Their use has often been restricted by the host-range of the cloning vectors used. However, the ecological relevance of these organisms in di¡erent environments will inevitably vary. It would therefore be advantageous to have di¡erent organisms to choose from when monitoring di¡erent environments [36]. It has also been shown that di¡erent organisms show great variations in sensitivity to di¡erent inducers of biosensor systems [37,38]. We demonstrate here the use of one construct by measuring the tetracycline concentration in milk from a cow treated with oxytetracycline. It was shown that detectable amounts of oxytetracycline are present in milk, even 5 days after treatment (Fig. 3). The biosensor technique shown here provides a sensitive, fast and cost-e¤cient alternative to standard procedures such as HPLC analysis of tetracyclines in milk [39,40]. We are currently using the biosensors to investigate gene transfer of plasmids carrying tetracycline resistance determinants, under the in£uence of various sub-lethal bioavailable tetracycline concentrations. Acknowledgements We thank Pia Windel Kringelum for technical assistance. This work was supported partly by the Danish Ministry of Food, Agriculture and Fisheries, Projects MIL 962 and MIL 96-3.
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Detection and quanti¢cation of tetracyclines by whole cell biosensors Lars Hestbjerg Hansen, SÖren Johannes SÖrensen * Department of General Microbiology, University of Copenhagen, SÖlvgade 83 H, DK-1307 Copenhagen K, Denmark Received 22 May 2000 ; received in revised form 12 July 2000; accepted 12 July 2000
Abstract Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing L-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different Gramnegative bacteria by conjugation. An Escherichia coli strain containing the Ptet -lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tetracycline ; Whole-cell biosensor; Reporter gene; lux; lacZ; gfp
1. Introduction In recent years, the use of biosensors to detect the presence of chemical compounds in complex environments has been widespread. Biosensors have enabled the detection and quanti¢cation of various compounds, such as metals [1], xenobiotic compounds [2], and antibiotics [3]. We have focussed on the detection and quanti¢cation of tetracycline by the use of biosensors based on the inducible resistance gene promoter Ptet (from Tn10) in combination with its regulatory gene tetR. This repressor/promoter interaction is well described elsewhere [4] as is its speci¢city in recognizing tetracyclines as inducers [5]. Biosensors based on fusions to this promoter will therefore produce a quantitative response to increasing levels of tetracyclines. Biosensors provide an inexpensive and sensitive alternative to non-microbiological procedures for detecting tetracyclines. These procedures include spectrophotometric methods [6,7] and high-performance liquid chromatography [8,9]. Most biosensor organisms used so far carry the biosensor gene cassettes on plasmids [3,10,11]. Problems with plasmid based systems can be varying copy numbers and loss of sensitivity [12]. It is also known that in some instances plasmids are lost from the organisms, if the organ-
isms are introduced to environments without any selective pressure [2]. The choice of reporter gene can vary, depending on the di¡erent environmental conditions in which the biosensors are used. For example, both gfp and lux will prove to be useless under anaerobic conditions since both folding of the GFP protein and light production from the lux genes require oxygen [13]. The gfp gene might be preferred in long-term incubations under low transcriptional activity, since the GFP is very stable and therefore accumulates within the cell [14]. We therefore developed a variety of £exible biosensor vectors for the detection and quanti¢cation of very low concentrations of tetracyclines in various environments. We present the choice of three di¡erent reporter gene systems: luxCDABE from Vibrio ¢scheri, lacZYA from Escherichia coli and gfp from Aequorea victoria, combined with the tetracycline inducible promoter from Tn10. Furthermore, we also transferred all three combinations into mini-Tn5 delivery vectors, which enable the introduction of the di¡erent biosensor-constructs as single chromosomal inserts into a variety of Gram-negative bacteria. 2. Materials, methods and results 2.1. Strains, plasmids and culture conditions
* Corresponding author. Tel. : +45 3532-2053; Fax: +45 3532-2040; E-mail : [email protected]
The E. coli strain MT102 was used as host strain in all
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 4 7 - 5
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DNA manipulation steps except for hosting the ¢nal biosensor constructs in the mini-Tn5 plasmids pUT-tetlux, -tetlac, -tetgfp. Because these plasmids need the Z protein for replication [24], they were transformed into MT102PIR. The MT102-PIR strains hosting the di¡erent biosensor plasmids were in turn used as donors in tri-parental ¢lter matings to insert the biosensor cassettes into the chromosomes of di¡erent Gram-negative bacteria. Strains of E. coli were grown in LB broth [25] at 37³C, unless otherwise stated. Other strains used were grown at 30³C. Tri-parental ¢lter matings were carried out as described earlier [16], and biosensor transconjugants were selected on basal salts medium [26] containing 0.2% glucose and 50-Wg ml31 kanamycin. All strains and plasmids are listed in Table 1. DNA plasmids and fragments were prepared, transformed, analyzed and manipulated by standard procedures [27]. Tetracycline^HCl was used in all inductions unless otherwise stated. 2.2. Cloning of the tetlux gene cassette The tet repressor/promoter was excised from pNK81 as a 1.6 kb BglII^EcoRI fragment containing tetR, Ptet and the beginning of the tetA gene. This fragment was then cloned into pUCD615, thereby creating an operon fusion between the tet-repressor, regulated promoter Ptet and the luxCDABE genes. This plasmid was called pUCDtetlux. The tetlux cassette could not be excised as one fragment. The lux genes were therefore excised as a SalI^PstI fragment and cloned into pLOW2. The plasmid was called pLOW2-lux. Next a SalI fragment from pUCD-tetlux
containing tetR and Ptet was ligated to SalI-digested pLOW2-lux. The resulting plasmid was named pLOW2tetlux. The entire tetlux construct was transferred from pLOW2-tetlux to pUT-kn-res as a NotI fragment to yield pUT-tetlux (Fig. 1A). 2.3. Cloning of the tetlac gene cassette The same 1.6 kb BglII^EcoRI fragment from pNK81 was ligated to BamHI^EcoRI digested pRS528, thereby creating an operon fusion between Ptet and the lacZYA genes. This plasmid was called pRS-tetlac. The newly created tetlac cassette was then transferred from pRS-tetlac to pLOW2 as a PstI^SalI fragment, yielding pLOW2-tetlac. This was done in order to generate NotI ends on the biosensor cassette. The cassette was then transferred from pLOW2-tetlac into the unique NotI site of the mini-Tn5 delivery vector pUT-kn-res to generate pUT-tetlac (Fig. 1B). 2.4. Cloning of the tetgfp gene cassette As a consequence of the incompatibility of cloning sites between the GFP vector pAG408 and the wild-type tet repressor/promoter fragment from Tn10, new restriction sites were introduced on the tet repressor/promoter fragment. This was done by PCR. The tet repressor/promoter was ampli¢ed from pNK81 using the following primers, Ptet NotI: 5P-ggcgaagcggccgctaagatctatg-3P and Ptet ClaI: 5Pagtggttatcgatatcttccgaagc-3P, to yield a 1126-bp fragment containing tetR, Ptet and the beginning of tetA. Thus, NotI
Table 1 Plasmids and strains Plasmids/strains Plasmids pAG408 pAG-tetgfp pLOW2 pLOW2-lux pLOW2-tetlac pLOW2-tetlux pNK81 pRB28 pRS528 pRS-tetlac pUCD615 pUCD-tetlux pUT-Kn-res pUT-tetgfp pUT-tetlac pUT-tetlux RK600 Strains MC4100 MC4100: :tetlac MT102 MT102-PIR NF1815
Replicon/species
Characteristics
Reference
R6K R6K p15A p15A p15A p15A pMB1 pMB1 pMB1 pMB1 pMB1 pMB1 R6K R6K R6K R6K pMB1
KmR , gfp KmR , Ptet -gfp KmR KmR , luxCDABE KmR , Ptet -lacZYA KmR , Ptet -luxCDABE AmpR , tetR-Ptet -tetA KmR , AmpR , luxCDABE AmpR , lacZYA AmpR , Ptet -lacZYA KmR KmR , Ptet -luxCDABE KmR , AmpR , mini-Tn5 pUT-Kn-res-Ptet -gfp pUT-Kn-res-Ptet -lacZYA pUT-Kn-res-Ptet -luxCDABE CmR
[15] This study [16] This study This study This study ATCC 77337 [17] [18] This study [19] This study [20] This study This study This study [21]
E. E. E. E. E.
glu+SmR MC4100: :Ptet -lacZYA leu pro thi SmR MT102: :pir-aphA p+ leu thi SmR
[22] This study [23] [16] N. Fiil
coli coli coli coli coli
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Fig. 1. Construction of the three biosensor cassettes and their insertion into pUT-Kn-res. (A) The tetlux cassette, an operon fusion between tetR-Ptet from Tn10 and the luxCDABE operon from V. ¢scherii, (B) the Ptet fusion to the E. coli lacZYA operon and (C) the fusion of Ptet to the gfp gene from A. victoria. Vertical striped bars represent the tetR gene, the cross-hatched bar represent part of cloning vector pRS528 and gray bars represent the truncated tetA gene from Tn10. Arrows indicate transcription start and restriction sites are indicated above the cassettes.
and ClaI sites (underlined) were generated at the ends of the tet repressor/promoter fragment. After a double digest using NotI and ClaI, the PCR fragment was ligated into pAG408, placing Ptet in front of the gfp gene. This construct was called pAG-tetgfp. The tetgfp cassette was transferred to pUT-kn-res by performing a second PCR. We used Ptet NotI as the forward primer and gfprevNotI: 5P-atggcggccgcattcattatttgt-3P as the reverse primer to generate a tetgfp fragment £anked by NotI sites (undelined). This construct was then ligated to the unique NotI site of pUT-kn-res to generate pUT-tetgfp (Fig. 1C). 2.5. Induction assays From an overnight LB culture of E. coli MT102-PIR/ pUT-tetlux, 1 ml was added to 40 ml of ABB1 medium [28] containing leucine and proline. 3-ml aliquots were
then transferred to disposable plastic tubes (for luminometer counting). Tetracycline was added to obtain various total concentrations as shown in Fig. 2A. The tubes were mixed by gentle inversion and incubated at room temperature without shaking. At various times, the emission of light per 30 s was measured in a BG-P Portable luminometer (MGM instruments, Hamden USA). Relative light units (RLU) at 50 min after incubation were then plotted against tetracycline concentration (Fig. 2A). An overnight culture of E. coli MT102-PIR/pUT-tetlac was diluted 100-fold in fresh LB medium containing 100 Wg ml31 ampicillin and various concentrations of tetracycline. The biosensor strains were grown for 3 h at 37³C with shaking. 0.5 ml was transferred to 1.5-ml Eppendorf tubes and placed on ice. 10 Wl of toluene was added to each tube and vortexed for 10 s. A L-galactosidase assay was then performed according to Miller [25]. The level of
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Fig. 2. The quantitative response of the three biosensor to increasing tetracycline concentrations. (A) Production of light from MT102-PIR/pUT-tetlux cells. (B) L-Galactosidase activity of MT102-PIR/pUT-tetlac cells. (C) Increasing £uorescence of MT102-PIR/pUT-tetgfp cells.
L-galactosidase was then plotted against tetracycline concentration (Fig. 2B). Finally, an overnight LB culture of E. coli MT102-PIR/ pUT-tetgfp was diluted 100-fold in fresh LB medium containing 100 Wg ml31 ampicillin and various concentrations of tetracycline, and grown overnight at 30³C for 16 h. A 3ml sample was washed twice and re-suspended in 0.9% NaCl (to minimize the background £uorescence of the LB medium). The 3-ml sample was then transferred to a cuvette and £uorescence was measured in a Luminescence Spectrometer LS 50B (Perkin Elmer, Buckinghamshire, UK). Excitation wavelength was 395 nm, emission was measured at 509 nm and relative £uorescent units were plotted against the concentration of tetracycline (Fig. 2C). 2.6. Use of an E. coli MC4100: :tetlac biosensor to measure bioavailable tetracycline in milk The pUT-tetlac construct was used to insert the tetlac biosensor cassette into the chromosome of E. coli MC4100 by performing a tri-parental ¢ltermating [16]. This strain was used to demonstrate the utility of these tetracycline biosensors. E. coli MC4100 with the tetlac cassette was used to determine the bioavailable tetracycline content in milk. Milk was taken from a cow treated with oxytetracycline. The treatment (5 mg kg31 ) was given intravenously at day 0, and samples of milk were taken twice daily (Fig. 3). Milk samples were centrifuged for 20 min and ¢ltered through a 0.2-Wm ¢lter. Di¡erent dilutions of milk-¢ltrate were added to test tubes containing 5 ml of LB with kanamycin (50 Wg ml31 ). To obtain a standard curve, known concentrations of oxytetracycline were also added to different tubes. The tubes were then inoculated with 50 Wl of E. coli MC4100: :tetlac taken from an overnight LB broth culture. The biosensor cells were grown for 3 h, toluenized and analyzed. Tetracycline concentrations in the milk samples were calculated by comparing the L-galactosidase ac-
tivity in milk samples with activities on the standard curve. Results were then plotted as oxytetracycline concentrations vs. sampling time (Fig. 3). 3. Discussion Unlike conventional methods for determination of concentrations of tetracyclines, the biosensors determine only the bio-available fraction of these compounds, that is, the actual concentration that the microbial community encounters in the environment. This method thereby does not measure the proportion of tetracyclines that is tightly bound to matrix material like soil particles or is in other ways biologically inactivated. This makes biosensors the tool of choice for investigating di¡erent substances, their concentration and their in£uence on and interaction with microbial communities. Furthermore, biosensors have the potential to become a cost-e¤cient and very sensitive alternative to conventional methods such as HPLC in determining concentrations of di¡erent compounds. It was clearly shown in this study that all three biosen-
Fig. 3. Oxytetracycline content in milk from a cow treated with 5 mg kg31 oxytetracycline. Tetracycline concentrations were measured using biosensor strain E. coli MC4100: :tetlac.
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sor constructs responded in a quantitative manner to different concentrations of tetracycline. We have constructed tetracycline biosensors containing a choice of reporter genes. Comparisons between the sensitivity of the di¡erent biosensors are impeded by the di¡erent assay growth conditions in this study. The need for separate assay conditions illustrates the di¡erences of the three reporters. We were unable to produce satisfactory induction of the lux genes during rapid growth. The amount of GFP present in our biosensors was insu¤cient with short incubations. Finally, both GFP and luciferase (from luxCDABE) failed to work well beyond 30³C. Consequently the assay conditions had to be adjusted for each reporter gene. The luxCDABE and lacZ genes are renowned for their sensitivity in biosensors [1,29,30], and the gfp is the reporter gene of choice for in situ microbial ecology [31,32]. Adding to the versatility of these biosensors is the fact that they can be conjugated into a variety of Gram-negative bacteria, where integration into the host chromosome will ensure a stable construct copy number and maintenance, even under non-selective conditions. We performed successful conjugations of the three biosensor mini-Tn5s to six di¡erent Gram-negative bacteria (data not shown). Until now, biosensor-constructs have been applied in a limited number of bacteria, such as E. coli [33], P. £uorescens [34] and P. putida [35]. Their use has often been restricted by the host-range of the cloning vectors used. However, the ecological relevance of these organisms in di¡erent environments will inevitably vary. It would therefore be advantageous to have di¡erent organisms to choose from when monitoring di¡erent environments [36]. It has also been shown that di¡erent organisms show great variations in sensitivity to di¡erent inducers of biosensor systems [37,38]. We demonstrate here the use of one construct by measuring the tetracycline concentration in milk from a cow treated with oxytetracycline. It was shown that detectable amounts of oxytetracycline are present in milk, even 5 days after treatment (Fig. 3). The biosensor technique shown here provides a sensitive, fast and cost-e¤cient alternative to standard procedures such as HPLC analysis of tetracyclines in milk [39,40]. We are currently using the biosensors to investigate gene transfer of plasmids carrying tetracycline resistance determinants, under the in£uence of various sub-lethal bioavailable tetracycline concentrations. Acknowledgements We thank Pia Windel Kringelum for technical assistance. This work was supported partly by the Danish Ministry of Food, Agriculture and Fisheries, Projects MIL 962 and MIL 96-3.
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