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Applied and Environmental Microbiology, September 2005, p. 5050-5055, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5050-5055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

Yung-Hun Yang,1 Hwang-Soo Joo,1 Kwangwon Lee,2 Kwang-Kyung Liou,3 Hei-Chan Lee,3 Jae-Kyung Sohng,3 and Byung-Gee Kim1,2*

School of Chemical and Biological Engineering,1 Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University,2 Institute of Biomolecule Reconstruction Lab, Sunmoon University, Seoul, Korea3

Received 19 January 2005/ Accepted 5 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
{gamma}-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
{gamma}-Butyrolactones are well recognized as bacterial hormone- and quorum-sensing molecules in Streptomyces, working in the concentration range of 10–8 to 10–9 molar (15), which can control cell density, secondary metabolism, and cell differentiation. After the structure (11, 23) and function (5, 20) of A-factor from Streptomyces griseus was elucidated, at least 17 kinds of similar molecules, such as SCB1 from Streptomyces coelicolor, factor I from Streptomyces viridochromogenes, IM-2 from Streptomyces lavendulae, and virginiae butanolides from Streptomyces virginiae, were identified, and their functions and structures were revealed by bioassay, gas chromatography-mass spectrometry, and nuclear magnetic resonance (6, 9).

The previous investigations demonstrated that the cell supernatants of some Streptomyces strains synthesizing butanolides with similar structures can have the same effect, but at different levels, on the antibiotic production and cell differentiation of the other strains, suggesting that the butanolides from one strain may interact with similar receptors in the other strains (7). In addition, the butanolides synthesized from one strain are not a single compound but are several compounds with different chiralities and/or different functional groups, such as hydroxyl and keto groups, suggesting that like gram-negative bacteria such as Pseudomonas aeruginosa, which produces dozens of 4-hydroxy-2-alkylqunolines (14), Streptomyces can also produce several similar butanolides. It would be very interesting to see why bacteria require several quorum-sensing molecules and what the functional roles of each molecule are. Too many things are still not known to answer these questions.

To answer the questions, we need to develop powerful tools to quickly identify such molecules and to harvest large amounts of the molecules for subsequent functional studies. As the butanolides contain one of two chiral centers, their chemical synthesis is not an easy task. Until now, a few butanolides had been chemically synthesized, and their functional roles were examined (15, 18, 21). Since, unlike N-acyl homoserine lactones from gram-negative bacteria, the butanolides from Streptomyces strains are produced in very small quantities, large volumes (i.e., from 300 liters to 1,200 liters) of the culture are required to accomplish nuclear magnetic resonance analysis as well as functional studies (21, 23). In addition, if the butanolides are heterogeneous isomeric mixtures, isolation of each fraction becomes even more difficult.

In recent proteomics studies, affinity capture technology has become quite popular and was often used to profile the structures of target molecules captured by the affinity tag. Based upon a similar concept, we thought that if the recombinant receptors of the butanolides, such as ArpA from S. griseus, FarA from S. lavendulae, BarA from S. virginiae, and SpbR from S. pristinaespiralis, were functioning properly in vitro (3, 12, 16, 17), then the receptors could be used as affinity capture molecules to harvest the butanolides, and subsequent mass spectrometric analysis may allow us to identify them. Then, all the quorum-sensing molecules which bind to the receptors could be easily profiled and collected by a simple treatment of the purified recombinant receptor molecules with cell supernatant solution. If this approach works, we can develop an efficient way to purify and identify many quorum-sensing molecules based on nucleotide sequence information for the receptor proteins.

Here we report a successful model system for the identification of SCB1 from S. coelicolor (Fig. 1). His-tagged ScbR overexpressed in Escherichia coli was harvested with Ni2+ metal affinity beads and subsequently used to isolate SCB1 from 100 ml of Streptomyces coelicolor culture broth. So far, this method appears to require the lowest volume of Streptomyces culture to collect a detectable amount of SCB1 molecules by electrospray tandem mass spectrometry (ESI-MS/MS). The method can be further applied to identify and prepare unknown quorum-sensing molecules for various receptor proteins.



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  		FIG. 1. Scheme for our current method. a) S. coelicolor culture in 250-ml flask. b) Ethyl acetate extraction. c) Overexpression of ScbR in E. coli. d) His tag purification and washing out of unbound proteins. e) Binding reaction between ScbR and SCB1. f) Ultrafiltration. g) Elution by pH change and boiling. h) ESI-MS/MS and bioassay.

 

   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains, plasmids, and media.
The bacterial strains and plasmids used in this work are listed in Table 1. Streptomyces coelicolor A3(2) M145 was obtained from the Korean Collection for Type Cultures (KCTC), and E. coli DH5{alpha} and BL21(DE3) were used as host strains. Streptomyces coelicolor A3(2) M145 was grown in R5– medium (10). Kanamycin and isopropyl 1-thio-ß-D-galactopyranoside (IPTG) were purchased from Sigma (Deisenhofen, Germany). All chemicals were of analytical grade.


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  		TABLE 1. Strains, plasmids, and primers

 
DNA manipulations and construction of plasmid.
The coding region of ScbR was amplified by PCR using primers (Table 1) from the genomic DNA of S. coelicolor A3(2) M145. The PCR amplifications were carried out for 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final stage at 60°C for 5 min using Pfu polymerase (Genenmed, Korea). The scbR fragments were digested with BamHI/HindIII (Roche, Germany) and inserted into the IPTG-inducible expression vector pET24ma, which was kindly donated by Hiroshi Sakamoto (Pasteur Institute, France). Other DNA manipulations, including preparation of plasmids, restriction enzyme digestion, ligation, and transformation of E. coli were done according to the methods of Sambrook et al. (19).

Expression and purification of ScbR.
pYH404 was introduced into E. coli BL21, and the transformants were grown in 200 ml of Luria-Bertani broth containing 50 mg liter–1 kanamycin at 37°C. When the cell optical density at 600 nm reached 0.6, 0.1 mM IPTG was added to the cell broth. The cells were harvested after 12 h of induction at 30°C. For the preparation of ScbR, the cells harvested from 200 ml of culture broth of recombinant E. coli BL21 were washed and suspended in 5 ml of 50 mM ammonium bicarbonate buffer (pH 7.5) containing 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.01% (vol/vol) 2-mercaptoethanol, and 1 mM dithiothreitol and then were subjected to ultrasonic disruption for 10 min. The supernatant solution was obtained after centrifugation (17,000 x g, 20 min).

For the purification of the His-tagged protein, the soluble protein was applied to Ni2+-nitrilotriacetic acid beads, which had been preequilibrated with base buffer (50 mM NaH2PO4, 0.05% NaCl, 0.05% Tween 20, pH 8.0). After the binding reaction proceeded for 2 h, unbound protein was removed by washing four times with the same buffer containing 20 mM imidazole. ScbR (9)-His tag was eluted three times from the column with 250 mM imidazole buffer. The product from each purification step was subsequently analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE).

Extraction of SCB1 from supernatant.
One hundred milliliters of culture broth of S. coelicolor A3(2) M145 grown in R5– medium for 72 h was extracted with an equal volume of ethyl acetate. The organic phase was then concentrated in a vacuum evaporator and was dissolved in 1 ml of 100% methanol in 1.5-ml Eppendorf tube. For the reaction with ScbR, the sample was evaporated once again in Speedvac and dissolved in 400 µl sterilized water.

Binding reaction with ScbR and SCB1.
Before the binding reaction, the solution of His tag-purified ScbR was washed three times with 10 ml of sterilized water to remove 250 mM imidazole in the buffer. Then, 10 ml of His tag-purified ScbR (0.15 mg/ml protein) was again centrifuged for further concentration with a 50-kDa-cutoff ultracentrifugal filter (Millipore, Ireland) for 1 h at 3,500 rpm, and 400 µl retentate was collected. The same volume (400 µl) of the concentrated SCB1 extraction solution was added to the 400 µl of the ScbR retentate and slowly shaken for 12 h at 25°C. The reaction mixture was subjected to ultrafiltration for 1 h at 3,500 rpm by adding three 10-ml portions of sterilized water, and then the pH of the mixture (800 µl) was changed to pH 2.0 by using HCl. The mixture was boiled for 3 min and centrifuged for 10 min. The supernatant was collected and evaporated to 20 µl with a Speedvac.

ESI-MS/MS analysis.
The mass spectrometric analysis in ESI-MS/MS was performed on an LCQ Deca XP ion trap mass spectrometer (Thermo Electron Corp.) with the nanospray source in positive-ion mode at a spray voltage of 1.75 kV. The heated capillary was maintained at a temperature of 200°C. For the MS/MS fragmentation, a 35 to 37% normalized collision energy and a 1.5-Da isolation width were used. The spray tip for the nanospray ion source was made by the method of Gatlin et al. (4) with a P-2000 laser puller (Shutter Instrument, Novato, CA) to create a 5-µm tip. Samples were infused to a C18 (Agilent)-packed spray tip by using a homemade high pressure bomb with a pressure of 1 MPa of nitrogen gas. The loaded sample was eluted isocratically with water-acetonitrile (50/50 [vol/vol]) at a flow rate of 0.3 µl/min. The maximum ion collection time was set to 50 ms, and three microscans were averaged per scan.

Bioassay with obtained molecules.
The biological activity was examined in liquid culture. For the preparation of the SCB1 sample, the extracted SCB1 was evaporated and dissolved in 500 µl of methanol, and 500 µl of filter-sterilized and distilled deionized water (0.2-µm filter; Satorious, Germany) was added to make the SCB1 sample in a 50% methanol solution. For the preparation of S. coelicolor cells, 0.5 µl of spore stock solution (7.92 x 105 colonies/µl) was cultured in 20 ml of R5– medium in a 250-ml baffled flask. To see the effect of SCB1 on cell growth and antibiotic production, 200 µl of the extracted SCB1 dissolved in 50% methanol was added to the starting 20 ml of culture medium.

Antibiotic extraction.
One milliliter of each sample was obtained at the specified times. After centrifugation in a microcentrifuge for 10 min, 900µl of supernatant was sampled for further analysis as described previously (1). Actinorhodin (Act) and undecylprodigiosin (Red) production was determined spectrometrically at 633 nm and 530 nm, respectively, by using 96-well plate and a multiscanner (Thermo Electron Corp., Finland) with 200µl of treated sample.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Overexpression and purification of ScbR.
The ScbR gene from S. coelicolor was cloned into pET24ma under the control of the T7 promoter, and all the sequences were confirmed by sequencing (GenoTech, Korea). ScbR was expressed in E. coli BL21. The overexpressed and His tag-purified ScbR of 27 kDa from the cell extract was confirmed by SDS-PAGE (Fig. 2). To avoid the formation of possible inclusion bodies, as in the case of ArpA (13), the culture was induced under mild conditions, i.e., 30°C and 0.1 mM IPTG. According to a previous report, recombinant FarA (rFarA), a recombinant receptor protein, tends to aggregate at high protein concentrations. Therefore, special care was needed to prevent aggregation during the course of purification and ultrafiltration of ScbR. In addition, to avoid any other unspecified spectra during ESI-MS/MS analysis, no glycerol or hemoglobin was added, which prevents rapid inactivation of ScbR at low or high concentrations, respectively (22).



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  		FIG. 2. SDS-12% PAGE of ScbR. a) His tag-purified ScbR. b) Protein standards.

 
Using Ni2+-nitrilotriacetic acid beads, soluble His-tagged ScbR was easily purified. His-tagged ScbR began to be eluted from the beads by 120 mM imidazole, but for complete elution from the beads, 250 mM imidazole buffer was used three times. After the His tag purification, to avoid the presence of a high concentration of imidazole in the solution, ultrafiltration was performed with water as described in Materials and Methods.

Capture of SCB1 by using ScbR.
A binding reaction was carried out between His tag-purified ScbR (3.75 mg/ml) solution and 400 µl of ethyl acetate solution containing butanolides extracted from the cell supernatant. The solution was slowly shaken for 12 h at pH 8.0 and 25°C. The same reaction conditions as for rFarA (22) were used.

For testing the feasibility of this experiment, a rough calculation was done on the ratio of the amounts of SCB1 in the cell supernatant to the purified recombinant ScbR. Taking into account the minimum effective concentration of SCB1 in the cell broth (21), 100 ml of the cell broth may contain about 125 to 250 nM of SCB1 (12 to 25 nmol), and/or 410 to 7,800 nM of the racemates (41 to 780 nmol) was expected to exist; 3.75 mg/ml of His tag-purified ScbR in 400 µl is about 60 mM (55 nmol protein). Assuming complete binding of SCB1 in the cell broth to ScbR and no loss of SCB1 during the extraction and concentration, the total amount of SCB1 reaches around 0.6 to 1.25 mM (12 to 25 nmol). Thus, despite all the significant losses during binding and elution caused by the instability of ScbR itself, the total amounts of SCB1 will easily exceed the detection limit of ESI-MS/MS.

As the binding affinity between receptor protein and {gamma}-butyrolactone changes according to pH change (17), a pH shift can be used for the elution of SCB1 from the ScbR-SCB1 complex. It was shown previously that IM-2 binding activity for rFarA is optimal at pH 8, but its binding activity is lost at below pH 5 (22). Thermal and acidic resistance of SCB1 has also been shown (21). Based upon this prior knowledge, to elute SCB1 from the ScbR-SCB1 complex, the pH of the reaction mixture was changed to pH 2 with HCl, and the mixture was subsequently boiled for 3 min to fully denature ScbR.

Identification of SCB1 and comparison with A-factor.
Prior to the detection of SCB1, A-factor, which is a similar quorum-sensing molecule for S. griseus, was analyzed by ESI-MS/MS as a control. Even though many studies on the quorum-sensing molecules of Streptomyces strains have been performed, no one has yet determined the MS/MS fragmented ion mass spectra of A-factor and SCB1. For the structural analysis of A-factor, MS/MS of A-factor (molecular weight, 242.2) was performed and characteristic peaks were assigned (Fig. 3a). Afterwards, the molecules captured by His-tagged ScbR were analyzed by MS/MS (Fig. 3b). Comparison of the MS/MS patterns of A-factor and the captured molecules revealed that the molecules captured by this method are certainly SCB1. The low-mass peaks, such as those at 81,109,127, and 199, are the peaks from the same lactone moiety of A-factor and SCB1, and the high-mass peaks, such as 207 (209 in Fig. 3b), 225 (227 in Fig. 3b), and 243 (245 in Fig. 3b) revealed the mass difference between hydroxy and ketone on the acyl chain (Table 2) (2). The same MS and MS/MS spectra of SCB1 were confirmed by using chemically synthesized SCB1 (data not shown).



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  		FIG. 3. Comparison of MS/MS with A-factor and SCB1. a) Authentic sample of A-factor from S. griseus. b) Captured molecules from S. coelicolor. Assignments of characteristic peaks are shown in Table 2.

 

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  		TABLE 2. Analyzed and expected ESI-MS/MS spectra of A-factor and SCB1

 
Bioassay by SCB1.
To further prove any biological effect of the putative SCB1 molecules captured by His-tagged ScbR on antibiotic production in S. coelicolor, a bioassay was performed by adding 200 µl of the captured putative SCB1 in 50% methanol solution to 200 ml of the S. coelicolor culture broth, as explained in Materials and Methods. Figure 4 shows the effect of captured molecules on antibiotic production. We can clearly see that the captured putative SCB1 molecules from the extract could effect early antibiotic production for both Act and Red (Fig. 4d and e). Although we cannot eliminate the possibility of an effect of other molecules in the extract on the antibiotic production, it is quite convincing that the molecules captured by His-tagged ScbR contain at least SCB1, which was confirmed by ESI-MS/MS. As a control experiment, His-tagged WecE transaminase from E. coli, which does not have anything to do with the affinity for SCB1, was prepared with the same procedure and used for the same binding and bioassay experiments. In this case, however, early antibiotic production was not observed in the culture broth, nor were any mass spectra of SCB1-like molecules detected, indirectly suggesting that the capture of SCB1 with His-tagged ScbR is protein specific (data not shown). Another thing to note from this experiment is that even though the added extract could elicit early antibiotic production, it did not increase the final yield of antibiotics, because of slower growth of the cell as well as early reduction in the cell mass compared with the case of no addition of captured SCB1.



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  		FIG. 4. Cell growth and antibiotic production curves. Two hundred microliters of the prepared reaction supernatant (see Materials and Methods) ({blacktriangledown}), 50% methanol filtered with a 0.2-µm sterilized filter ({circ}), or sterilized water (•) was added to the culture broth. a) Wet cell mass. b) Red production. c) Act production. d) Red production per milligram of wet cell mass. e) Act production per milligram of wet cell mass. O.D., optical density; AU, arbitrary units.

 
To definitely confirm that the captured molecule is SCB1, chemically synthesized SCB1 and VB-C racemates were analyzed by mass spectrometry (data not shown) and applied. After 65 h, only the addition of chemically synthesized SCB1 and the captured fraction showed the early antibiotic production (Fig. 5). This result also suggests that the captured molecule would be mainly SCB1 and that our method of using His-tagged ScbR functions successfully for capture of quorum-sensing molecule like SCB1.



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  		FIG. 5. Comparison of early antibiotic production by different quorum-sensing molecules. Two milliliters of R5– medium and 10 µl of prepared sample were used. a) Distilled water only. b) 50% methanol. c) VB-C racemates in 50% methanol. d) SCB1 racemates in 50% methanol. e) Captured putative SCB1 in 50% methanol.

 
The above ESI-MS/MS and bioassay data showed that an affinity capture method for quorum-sensing molecules could be developed using a very small amount of culture broth. With this method, only genomic information on receptor genes is required to identify and harvest quorum-sensing molecules. The next questions would be whether or not all the receptor proteins (CprA and CprB) found for SCB1 in S. coelicolor also display similar binding affinity for SCB1 and whether the receptor binding is specific for SCB1-like molecules, as several different SCB1-like molecules are found in the culture broth. Our approach could be a new "pathfinder" to find possible quorum-sensing molecules quite easily and efficiently.


   ACKNOWLEDGMENTS
 
This work was partially supported by the IMT-2000 program (Korean Ministry of Commerce) and Nano Bioelectronics and Systems ERC (Korean Ministry of Science and Technology).

We thank S. Horinouchi for generous provision of A-factor, which played a key role for the mass spectrometric analysis. We are also grateful to GeneChem Inc. (Taejon, Korea) for the supply of SCB1 and VB-C racemates.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Chemical and Biological Engineering, Seoul National University, Shillim Dong, San 56-1, Kwan-ak Gu, Seoul, Korea. Phone: 82-2-880-6774. Fax: 82-2-873-6020. E-mail: byungkim{at}snu.ac.kr. Back


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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, September 2005, p. 5050-5055, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5050-5055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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