A Salmonella Detection System Using an Engineered DNA Binding

May 6, 2000 - PCR products were visualized by submarine gel electrophoresis on 3 % agarose gel in TBE buffer.7. Measurement of the Total DNA in the PC...
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Anal. Chem. 2000, 72, 2809-2813

A Salmonella Detection System Using an Engineered DNA Binding Protein That Specifically Captured a DNA Sequence Akira Takeuchi*,† and Koji Sode‡

R & D Division, Q. P. Corporation, 5-13-1, Sumiyoshi-cho, Fuchu-shi, Tokyo 183-0034, Japan, and Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganeishi, Tokyo 184-8588, Japan

We have developed a novel method for the detection with high selectivity of a double-stranded DNA fragment using an engineered DNA-binding protein, DnaA IV, a fusion protein of the DNA-binding domain of DnaA and glutathione S-transferase. The DNA fragment detection system is based on DNA-protein interaction and consists of sequence-specific binding of DnaA IV with a DNA fragment containing the DnaA box. DnaA IV, while not capturing other DNA fragments, specifically captured that containing the DnaA box. Because the oriC fragment containing the DnaA box could be specifically amplified by PCR from the genus Salmonella, the DNA fragment detection system was adapted for the detection of Salmonella. The Salmonella detection system using PCR amplification and the engineered DNA-binding protein could distinguish 104 cfu/mL Salmonella from 106 cfu/ mL contaminating bacteria. The development of rapid, sensitive, and accurate DNA-sensing systems has become a necessity in the field of clinical analyses and food quality control. Recent advances in the development of DNA arrays, which enable us to screen and analyze a genomic database, are particularly useful for drug discovery. However, the current market prices of these technologies are limiting. Therefore, for routine clinical analyses and food quality control based on DNA sensing, the analyses of PCR-amplified products are still in high demand. In order to analyze the PCR-amplified product against specific primers and targets, confirmation of the amplified sequence is necessary, considering the existence also of nonspecific amplification products. Recently, several methods, such as molecular beacon,1 TaqMan chemistry,2 and LightCycler,3 based on DNADNA hybridization and fluorescence resonance energy transfer (FRET), were introduced. In the methods using FRET probes, amplification and detection were simultaneous, so that PCR * Corresponding author: (tel) +42-3615190; (fax) +42-3616271. † Q. P. Corporation. ‡ Tokyo University of Agriculture and Technology. (1) Piatek, A. S.; Tyagi, S.; Pol, A. C.; Telenti, A.; Miller, L. P.; Klamer, F. R.; Alland, D. Nat. Biotechnol. 1998, 16, 359-63. (2) Sharma, V. K.; Dean-Nystrom, E. A.; Casey, T. A. Mol. Cell. Probes 1999, 4, 291-302. (3) Bernard, P. S.; Pritham, G. H.; Wittwer, C. T. Anal. Biochem. 1999, 273, 221-8. 10.1021/ac991232n CCC: $19.00 Published on Web 05/06/2000

© 2000 American Chemical Society

amplification can be monitored in real time. These methods are, however, based on the detection of single-stranded target DNA molecules hybridizing with DNA oligonucleotides labeled with fluorescent probes, and therefore, the heat denaturation step and annealing step are necessary for the detection. Specific DNA detection of a double-stranded structure can be achieved without the denaturation step using DNA-binding proteins. Stoller et al. reported that two mouse monoclonal antibodies recognized different sites on the Z-DNA4 and also that a single-chain Fv of anti-DNA antibody could be expressed in large amounts.5 However, the application of DNA-specific antibodies for routine measurements is not practical considering their cost, the rigidity with which the antibodies identifies the sequence, and the availability of the specific antibody for the target DNA sequence. DNA detection using an engineered DNA-binding protein could also be easily applied to living cells. Patterson et al. described quantitative monitoring of the localization and expression using a fusion protein of the TATA-binding protein and green fluorescent protein in living yeast cells.6 Considering that the direct detection of a double-stranded DNA fragment using a DNAbinding protein is possible, further screening and investigation of sequence-specific DNA-binding proteins will expand the potential applications of this method. In addition, we have been engaged in the development of a technique for highly sensitive and accurate detection of Salmonella from food products, using Salmonella-specific DNA primers for PCR amplification. This method is based on the amplification of the oriC gene, by which genus Salmonella can be selectively detected and distinguished from other foodborne microorganisms, including various biochemically similar enteric bacteria, such as Escherichia coli and Citrobacter sp.7 In order to detect the amplified Salmonella oriC gene selectively, we focused on oriC-gene-specific binding protein, DnaA. The DnaA protein, an initiator protein encoded by the dnaA gene binds to the oriC sequence, the replication origin of chromosomal DNA, which contains four (R1, R2, R3, R4)8 or five (R1-R4, M)9 9-bp repeats (DnaA box). (4) Polymenis, M.; Brigido, M. M.; Sanford, D. G.; Stollar B. D. Biotechnol. Appl. Biochem. 1993, 18, 175-83. (5) Stollar, B. D. Methods: A Companion to Methods in Enzymol. 1997, 11, 12-9. (6) Patterson, G. H.; Schroeder, S. C.; Bai, Y.; Weil, A.; Piston, D. W. Yeast 1998, 14, 813-25. (7) Takeuchi, A.; Sode, K. Biotechnol. Tech. 1999, 13, 81-5. (8) Fuller, R.S.; Funnell, B.E.; Kornberg, A. Cell 1986, 38, 889-900.

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The DnaA protein is highly conserved among different bacteria.10 Its amino acid sequence of Salmonella typhimurium closely resembles that of E. coli (single mismatch in GenBank data). The Salmonella dnaA gene compliments E. coli dnaA (Ts) strains and the properties of the Salmonella and E. coli replication proteins are very similar.11 The DnaA protein is subdivided into four domains. Domain IV, the 94 C-terminal amino acids of the DnaA of E. coli, was reported to be required and sufficient for binding to the DNA fragment containing the oriC gene.12 Majka et al. showed that the immobilized domain IV of DnaA of E. coli could recognize and remove the DNA fragment containing the oriC gene.13 A total of 308 DnaA boxes of the stringent definition (5′TTA/TTNCAC/AA-3′) were found on the E. coli genome, but the DnaA protein bound with unusually high affinity to only 6 regions.14 Therefore, the application of DnaA, or the protein region of DnaA containing the DNA-binding domain, is expected to be utilized for the detection specifically and selectively of the doublestranded DNA fragment containing oriC or the region containing the DnaA boxes, even in the presence of many nonspecific DNA fragments without the denaturation step. In this study, we report a novel DNA detection system using a partial sequence of DnaA combined with PCR amplification of the oriC region of Salmonella, to detect the presence of Salmonella. EXPERIMENTAL SECTION Strains and Media. Citrobacter freundii IF012681 and TLQ11153 (foodborne), Enterobacter cloacae IF03320, E. coli ATCC10536 and ATCC12042, Erwinia carotovora ATCC15713, Proteus morganii IFO3168, Proteus vulgaris IFO3851, Salmonella derby ATCC6960, Salmonella enteritidis IF03313 and TLQ11129 (foodborne), Salmonella minnesota ATCC9700, Salmonella montevideo ATCC8387, Salmonella oranienburg ATCC9239, Salmonella pullorum ATCC9120, Salmonella senftenberg ATCC8400, and S. typhimurium IFO12529 were streaked on standard plate count (SPC; Eiken Co., Tokyo, Japan) plates and cultured overnight at 35 °C. The growth conditions of E. coli DH5R and its derivatives are shown below. Expression of DnaA IV. PCR primer sets (Forward: 5′CCggATCCATgAAAAAAgCggACgAAAATgATATTCgT-3′. Reverse: 5′-CCgAATTC1TACgATgACAATgTTCTgATTAAATFCgA3′) were designed based on the dnaA sequence of S. typhimurium15 and the amplified DNA-binding domain and its flanking sequence (C-terminus of 161 amino acids) from S. enteritidis TLQ11129, using 2.5 units of rTaq (TaKaRa) in 50 µL of the recommended buffer in 35 cycles, each cycle consisting of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min. The PCR product was purified on glass milk (Geneclean II, BiolO1), digested with BamHI and EcoRI (TaKaRa), ligated to the BamHI-EcoRI-opened pGEX-2T (9) Matsui, M.; Oka, A.; Takanami, M.; Yasuda, S.; Hirota, Y. J. Mol. Biol. 1985, 184, 529-33. (10) Fujita, M. Q.; Yoshikawa, H.; Ogasawara, N. Gene 1992, 110, 17-23. (11) Roth, A.; Messer, W. EMBO J. 1995, 14, 2106-11. (12) Maurer, R.; Osmond, B. C.; Shekhtman, E.; Wong, A.; Botstein, D. Genetics 1984, 108, 1-23. (13) Majka, J.; Jakimowicz, D.; Zarko-Postawka, M.; Zakrzewska-Czerwinska, J. Nucleic Acids Res. 1997, 25, 2537-8. (14) Roth, A.; Messer, W. Mol. Microbiol. 1998, 28, 395-401. (15) Skovgaard, O.; Hansen, F. G., J. Bacteriol. 1987, 169, 3976-81.

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vector, transformed into E. coli DH5R to obtains E. coli TLQ11275. Cells of E. coli TLQ11275 were cultured in 1 L of LB medium at 35 °C to OD600 ) 0.6 and induced with 1 mM IPTG for 3 h. The cells were harvested, resuspended in 25 mL of PBS (140 mM NaCI, 2.7 mM KC1, 10 mM Na2HPO4, 1.8 mM KH2PO4), sonicated, and centrifuged at 10 000g. The crude extract was sedimented with 40% (saturated) Na2SO4, resuspended in 25 mL of PBS, desalted by Sephadex G25 (PD10, Amersham Pharmacia Biotech), and adsorbed onto l mL of Glutathione Sepharose 4B gel (Amersham Pharmacia Biotech) overnight at 5 °C. The gel was washed 5 times with PBS, rinsed with binding buffer (50 mM Tris, 70 mM KCI, 1 mM EDTA, 1 mM mercaptoethanol, 10 % (v/v) glycerol (for fluoroassay, Kanto Chemicals, Tokyo, Japan), pH7.2) and used as DnaA IV. The concentration of DnaA IV was measured using GST 96-well Detection Module (Amersham Pharmacia Biotech). Amplification of oriC or Nonspecific Fragments. About 107 or 109 cfu of Salmonella or non-Salmonella strain were resuspended in 1 mL of distilled water, boiled for 10 min, centrifuged at 10 000g and used as the DNA template for the amplification of oriC or nonspecific fragments. Amplification of the oriC fragment was carried out as described previously.7 Using the heat extract of 109 cfu of Citrobacter sp. TLQ11153 as the template, nonspecific fragments were amplified. Using the heat extract of DNA from ∼107 cfu/mL suspension of E. coli. E92812 7 as the DNA template, STa (5′-TAATAGCACCCGGTACAAGC-3′) and STb (5′-ATAAAAGTGGTCCTGAAAGC-3′) as the PCR primers,16 the ST fragment, a portion of the st gene that codes for a heat-stable enterotoxin, was amplified in 30 reaction cycles (each cycle consisting of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min). The ST fragment did not exhibit any homology with oriC (80 bp). The PCR products were visualized by submarine gel electrophoresis on 3 % agarose gel in TBE buffer.7 Measurement of the Total DNA in the PCR Mixture. One microliter of the PCR mixture, 0.1 µL of SyBR Green I (Molecular Probes, Leiden, The Netherlands), and 1 µL of PBS were mixed and held for 10 min in a dark place before the fluorescence was measured (excition 470 nm, emission 530 nm). A standard curve was constructed using the φxl74/HaeIII digest (Toyobo, Osaka, Japan). Dilution buffer (1× PCR buffer + dNTPs) was used as the control. Recovery of DNA Captured by DnaA IV. DNA fragments (1-10 ng/µL oriC and 1-10 ng/µL ST fragment in 30 µL of binding buffer) were mixed with 30 µL of DnaA IV (280 ng of DnaA IV/µL of Glutathione Sepharose 4B gel) and incubated for 2 h at room temperature. The supernatant with the unbound material was removed by centrifugation. The DnaA IV with the DNA fragment was washed twice with binding buffer, rinsed with PBS, and suspended in 30 µL of the elution buffer (2.5 M NaC1, 50 mM Tris, pH 8.0), and the DNA fragment was washed off for 10 min at room temperature. Using a cosedimenter (Roger Rapid, Biodelta, Oeyhausen, The Netherlands), the DNA fragment was precipitated with ethanol, followed by agarose gel electrophoresis. Measurement of the DNA Captured by DnaA IV. DNA fragments (1-10 ng/µL oriC and/or 1-10 ng/µL nonspecific fragments in 30 µL of binding buffer) or dilution buffer was mixed with 30 µL of DnaA IV (280 ng of DnaA IV/µL Glutathione (16) Enami, H.; Tanaka, O.; Nakatani, S. Japanese Patent H3-112499, 1991.

Figure 2. Quantitative detection of oriC fragment using DnaA IV. OriC fragment or a nonspecific fragment was mixed and captured by DnaA IV. The DNA fragment not captured by DnaA IV was removed, SyBR green I staining was performed, and the fluorescence change was measured (ex ) 470 nm, em ) 530 nm). Average values and errors of triplicate trials are presented. Key: (O) oriC fragment, (b) nonspecific fragment, (2) ST fragment, and (9) φx174/HaeIII digest. Figure 1. Scheme of detection of oriC fragment.

Sepharose 4B gel) and incubated for 2 h at room temperature. The supernatant with the unbound material was removed by centrifugation. The DnaA IV with the DNA fragment was washed 5 times with binding buffer and resuspended in 1 mL of PBS. The suspension was mixed with 1 µL of SyBR Green I, before the fluorescence was measured. Then, the change in fluorescence (∆F value) was calculated.

∆F value ) fluorescence (oriC and/or nonspecific fragment) - fluorescence (dilution buffer) Detection of the oriC Fragment of Salmonella. S. enteritidis IF03313 cells (0.12 cfu/mL) were spiked into buffered peptone water (BPW broth, Oxoid, Hampshire, U.K.) and cultured at 35 °C. The culture was sampled and spread on xylose lysine desoxycholate (XLD, Wako, Osaka, Japan), and the black colonies were counted as Salmonella. At each sampling time, the culture was heat-extracted for DNA and the extract was used as PCR template. RESULTS AND DISCUSSION Scheme of Detection of the oriC Fragment. Figure 1 shows the scheme of detection of the oriC fragment using DnaA IV. Using PCR, the oriC fragment of Salmonella could be amplified, genus specifically.7 However, this PCR product may also contains nonspecific DNA fragments. When DnaA IV-affinity gel was mixed with the DNAs amplified by PCR, DnaA IV was found to bind with the dnaA box in the oriC fragment. After removal of nonspecific fragments by sedimentation and staining with intercalator, the oriC fragment could be detected by the change in fluorescence.

Preparation of Immobilized DnaA IV. The DNA-binding domain and its flanking sequence of S. enteritidis TLQ11129 was amplified and inserted into the GST fusion protein expression vector. The amino acid sequence determined by the sequence analysis of the amplified and inserted DNA fragments revealed that the DnaA sequence of S. enteritidis TLQ11129 was identical to that of S.typhimurium (complete agreement in 161 amino acids of the C-terminus) and closely resembled that of the E. coli (single mismatch) DnaA (data not shown). The overexpressed GST-DnaA fusion protein was purified by Glutathione Sepharose 4B gel (DnaA IV). The Mw of recombinant DnaA IV on SDS polyacrylamide gel was 46 kDa and agreed well with the value calculated from the amino acid sequence. Incubation of the DnaA IV prepared thus with the oriC DNA fragment amplified from genomic DNA of S. enteritidis resulted in a gel shift corresponding to migration of the oriC DNA fragment. However, no gel shift was observed when a control DNA fragment, ST in this case, which does not contain a DnaA box, was incubated with DnaA IV. Therefore, DnaA IV retained its selective binding ability with the oriC region. Detection of the Amplified oriC Region Using DnaA IV. DnaA IV was immobilized on Glutathione Sepharose 4B gel, and selective detection of the oriC gene was attempted. Using the heat extract of 107 cfu/mL S. enteritidis TLQ11129 and 109 cfu/mL Citrobacter sp. TLQ11153 as templates, the oriC or the nonspecific fragment was amplified in 30 or 35 cycles and yielded a 140-bp or other bands of DNA fragments, respectively. The total DNA in the PCR-amplified mixture was measured with SyBR Green I, diluted to 0.5-5.0 ng of DNA/mL, and mixed with DnaA IV. The DNA fragment that was not captured by DnaA IV was removed and stained with SyBR Green I (Figure 2). Because the oriC fragment amplified from Salmonella was captured by DnaA IV, a significant increase in SyGR green I fluorescence (∆F value larger Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

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Figure 4. Salmonella detection using PCR amplification of the oriC fragment and DnaA IV. S. enteritidis IF03313 cells were spiked into BPW broth and cultured at 35 °C. Cultures were sampled at various time points, and the numbers of viable cells was counted on the XLD plate. Finally, the cultures were heat-extracted for the DNA by 10min boiling. and the extract was used as the PCR template. Measurement of DNA concentration, fluorescence change, and electrophoresis were performed by the same procedure used in Figure 3. Lane number is the same as the culture time. Lane M, φxl74/HaeIII digest.

Figure 3. Differentiation of oriC fragment from nonspecific fragment by DnaA IV. Cell suspensions of each strain were boiled for 10 min, and the extracts were used as the DNA template for PCR amplification. (A) The DNA fragments in the PCR product were stained with SyBR green I, and the total DNA concentration was quantified. Then, the PCR products were mixed and those captured by DnaA IV assessed. The DNA fragments not bound to the DnaA IV were removed, SyBR green I staining was performed, and the fluorescence change was measured (ex ) 470 nm, em ) 530 nm). (B) The PCR products were subjected to 3% agarose gel electrophoresis. The lane number corresponds to the number in (A). Lane M, φx174/HaeIII digest.

than 100) was observed. The ∆F value increased quantitatively according to the amount of oriC fragment present, between 0.5 and 3.0 ng/µL. However, the nonspecific fragment amplified from Citrobacter was captured poorly; therefore, this sample showed almost no signal. The ST fragment and φx174/HaeIII digest, which did not have a DnaA box, also were not captured by DnaA IV. The fluorescence of a mixture of oriC fragment (0.5-3.0 ng of DNA/µL) and ST fragment (5.0 ng of DNA/µL) also increased according to the content of the oriC fragment and agreed well with the value obtained without the ST fraction (data not shown). Sequence-specific capture of the oriC fragment by DnaA IV was observed and quantitative detection of the oriC fragment prepared from Salmonella in the presence of contaminated DNA was performed. Genus-Specific Detection of Salmonella Using SequenceSpecific Detection of the oriC Fragment. We attempted application of the DnaA IV-based DNA detection method for the detection of PCR-amplified products containing nonspecific amplified samples. The results are summarized in Figure 3. The PCR amplification of non-Salmonella strains did not yield the 141 bp oriC bands. However, when the heat-extracted culture of Citrobacter sp. TLQ11153 was used as the template, a clear band with larger Mw was obtained. Such amplification of a nonspecific DNA 2812 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

fragment was also observed when E. coli ATCC10536 was used as the template. These PCR products (∼3 ng/µL) from nonSalmonella strains were subjected to the DnaA IV-based assay. Although the presence of the oriC DNA fragment resulted in a significant increase in fluorescence, these samples showed almost no signal. Therefore, this method can detect the amplified DNA fragment specifically and also distinguish the nonspecifically amplified DNA fragment based on the internal DNA sequence. Next, we attempted to evaluate the detection threshold of this method. BPW broth is widely used for the detection of Salmonella in food samples. S. enteritidis IF03313 cells were cultured at 35 °C in BPW broth, and the culture was subjected to PCR amplification followed by DnaA IV-based assay (Figure 4A). Increase in fluorescence was observed from the samples after more than 8 h of incubation. These results showed that the oriC fragment was amplified by PCR. We confirmed amplification of the oriC fragment by gel electrophoresis (Figure 4B). The results indicated that the oriC fragment, amplified by PCR, could be detected without using electrophoresis and indicated that Salmonella was detectable by employing the system using PCR amplification and an engineered DNA-binding protein. For each sampling time, the culture was heat-extracted and the DNA was used as the PCR template (5 µL of heat extract/ PCR tube). Of the samples incubated for more than 8 h, the cell concentration of Salmonella was in excess of 104 cfu/mL. The PCR template contained over 50 copies of the Salmonella genome from which the oriC fragment could be amplified. The minimum time and cell concentration required for the Salmonella detection system using PCR amplification and the engineered DNA binding protein were ∼6 h (3 h for PCR amplification and 3 h for detection using DnaA IV) and 104 cfu/mL culture, respectively. The Salmonella detection system using PCR amplification and the engineered DNA-binding protein has the advantage that it does not need heat denaturation and offers the potential for homogeneous assay of double-stranded DNA with a specific sequence.

CONCLUSIONS The DNA fragment detection system, which is based on sequence-specific binding between DnaA IV, a fusion protein of the DNA-binding domain of DnaA and glutathione S-transferase, and a DNA fragment containing DnaA box was constructed. Because DnaA IV captured the DnaA box in the oriC fragment, the double-strand DNA detection system using DnaA IV could be applied for Salmonella detection using PCR amplification. The DnaA box sequence in the oriC gene is well conserved in almost all the bacteria.17 However, the oriC gene sequence, except for the DnaA box sequence, is otherwise not conserved and the (17) Zakrzewska-Czerwinska, J.; Majka, J.; Schrempf, H. J. Bacteriol. 1995, 177, 4765-71.

genus can be distinguished specifically using a primer set designed on the basis of nonconserved regions.7 Therefore, the oriC fragment containing the DnaA box can amplified by PCR from the organism specifically; subsequently, the amplified DNA fragment can be detected by DnaA IV. ACKNOWLEDGMENT The authors are grateful to Dr. Wakako Tsugawa for her helpful discussion and advice. Received for review October 29, 1999. Accepted February 17, 2000. AC991232N

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