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Autonomously Replicating Plasmid Transforms Sorangium cellulosum So ce90 and Induces Expression of Green Fluorescent Protein
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 5, 385–390. 2007 DOI: 10.1263/jbb.104.385
© 2007, The Society for Biotechnology, Japan
Autonomously Replicating Plasmid Transforms Sorangium cellulosum So ce90 and Induces Expression of Green Fluorescent Protein Yun Tu,1 Guo-Ping Chen,1 and Yuan-Liang Wang1* College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China1 Received 30 March 2007/Accepted 3 August 2007
A recombinant plasmid, pRP-GFP, harboring the green fluorescent protein (GFP) gene from the broad-host-range mobilizable plasmid pRK415 of the RK2 family was constructed and transferred by conjugation from Escherichia coli S17-1 to Sorangium cellulosum So ce90. The results of Southern blot analysis showed that the plasmid replicated autonomously without being integrated into the chromosome of the bacterium. In pRP-GFP, gfp was driven by a So ce90 DNA fragment called EpoPro, which is an 890-bp DNA segment spanning from −890 to −1 relative to the start codon (GTG) of epoA, a gene that encodes EPOS A, which is presumed to be involved in the initiation of epothilone biosynthesis. The GFP in the transformed S. cellulosum So ce90 was detected by fluorescence microscopy, which suggests that the EpoPro fragment had the function of promoter. Because the green fluorescent bacilli could be directly observed by fluorescence microscopy, the stable expression of GFP was rapid and convenient for conjugation screening in addition to the antibiotic resistance genes within the constructed plasmid. This is the first report on an exogenous plasmid that can be stably maintained as an extrachromosomal element in S. cellulosum. [Key words: Sorangium cellulosum, autonomously replicating plasmid, green fluorescent protein, conjugation, epothilone]
into the chromosome of the host in such transfer systems. Nevertheless, for homology recombination, integrating DNA with regions of homology of less than 1000 bp could be extremely difficult because the recombination frequency in the host is very low (9). Julien and Fehd (8) showed that the transposition of a transposon into So ce90 is rare (1 in 10,000 to 1 in 100,000). So far, no endogenous plasmid had been found in S. cellulosum, and few attempts to transfer an autonomously replicating exogenous plasmid into this species have been reported (12). Such plasmid would be of great value for genetic research using bacteria. In this paper, we constructed a plasmid, pRP-GFP, that can be transferred to So ce90 easily and replicated autonomously without being integrated into the chromosome of S. cellulosum. Our results indicate that the plasmid is a fastacting, convenient and valuable genetic tool for research on So ce90. Furthermore, epothilone biosynthetic gene clusters are made up of seven genes transcribed in the same direction, encoding five polyketide synthases (PKSs) (epoA-epoE), a nonribosomal peptide synthetase (NRPS) (epoP) and a cytochrome P450 (epoF) (13). Although the gene cluster has been sequenced, the promoter of this operon is still unknown. In this study, we found that the fragment EpoPro, which was used to construct pRP-GFP, spanning from −890 to −1 relative to the start codon (GTG) of epoA, could drive the expression of gfp in S. cellulosum. It is suggested that this fragment has the function of a promoter.
The gram-negative bacterium Sorangium cellulosum belongs to the order Myxococcales, which is composed of soildwelling bacteria with complex social behaviors and multicellular developmental processes (1). Over the past 20 years, S. cellulosum have become well known for their secondary metabolites with unique structures and biological activities, e.g., epothilones (2), soraphens (3), disorazols, and chivosazoles (4). Of these bioactive products, epothilones have attracted considerable attention as a new class of active microtubule-stabilising cytotoxic agents for the treatment of solid tumors by means of the same mechanism of action as that of Taxol (2). Above all, epothilones have more advantages than Taxol, such as better water solubility, simple structures and the activity of the P-glycoprotein (5). The marked potential for epothilones makes Sorangium species very interesting test subjects for molecular biological studies of genetic control and molecular mechanisms. Of the series of Sorangium strains that have been isolated, So ce90 has received the most attention because this strain was shown to excrete more than 30 different epothilones (6). However, Sorangium strains are some of the most difficult myxobacteria to work with. They have the longest doubling time of any myxobacterium, up to 16 h, and very few genetic tools are available (7, 8). Even now, conjugation in S. cellulosum has been established to introduce DNA into the bacterium through homology recombination or transposons (8–11). All the results show that foreign DNA is integrated * Corresponding author. e-mail: [email protected] phone/fax: +86-23-65102508 385
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MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions The strains and plasmids used in this study are listed in Table 1. A seed culture of S. cellulosum So ce90 (DSM 6773) was obtained from the German Collection of Microorganisms and Cell Cultures. It was grown at 30°C in liquid G52 medium and plated on VY/2 agar plates as described by Gerth et al. (2). GY medium was used for conjugation. It contained 2 g/l glucose, 2 g/l yeast extract, 8 mg/l Na-FeIII-EDTA, 1 g/l MgSO4 ⋅7H2O, 1 g/l CaCl2 ⋅2H2O and 11.5 g/l HEPES. The medium was adjusted to pH 7.4 with KOH and autoclaved for 30 min at 121°C. If required, the media were supplemented with 50 µg/ml tetracycline (Sigma, St. Louis, MO, USA) and 10 µg/ml kanamycin sulfate (Sigma). Escherichia coli strains JM109 and S17-1 were used for plasmid subcloning and conjugation, respectively. E. coli strains were grown in LB medium at 37°C. When necessary, antibiotics were used at the following concentrations: 50 µg/ml ampicillin (Sigma), 50 µg/ml kanamycin sulfate, and 50 µg/ml tetracycline. E. coli S17-1 with a plasmid was used as a conjugation donor. DNA preparation S. cellulosum So ce90 genomic DNA was isolated using the Genomic DNA Purification kit (BioDev, Beijing, China) according to the manufacturer’s protocol. Plasmid DNA purification was carried out using E.Z.N.A Plasmid Miniprep kit I (Omega, Doraville, GA, USA) according to the manufacturer’s protocol. Construction of expression plasmid pRP-GFP All the restriction enzymes used, Taq polymerase and T4 DNA ligase were purchased from Takara (Dalian, Liaoning, China) and used according to the manufacturer’s recommendations. The PCR products were directly ligated into pMD18-T using the pMD18-T vector kit (Takara) according to the manufacture’s protocol and sequenced by Invitrogen Biotechnology (Shanghai, China). The pMD18-T vector, a specific TA cloning plasmid for cloning of PCR products, was rebuilt from pUC18 by Takara company. The promoterless GFP DNA segment was amplified from the plasmid pGFP2.1, using the forward primer containing a PstI site and the ATG initiation codon (GFP-F: 5′-CTGCAGATGGGTAGT AAAGGAGAAG-3′) and the reverse primer containing a KpnI site (GFP-R: 5′-GGTACCCTATTTGTATAGTTCATCC-3′). The conditions used for the PCR reaction were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. The 726-bp GFP fragment was ligated into the plasmid pMD18-T to
create pMD18-GFP. The 890-bp EpoPro fragment, spanning from −890 to −1 relative to the start codon (GTG) of epoA was PCR amplified from S. cellulosum So ce90 DNA with the forward primer containing a HindIII site (Pro1F: 5′-AAGCTTCCTGCCTCGATGGCCTTCCC3′) and the reverse primer containing a PstI site (Pro1R: 5′-CTGC AGGACGGGCACATCCTCAGCG-3′), and then ligated into the plasmid pMD18-T to create pMD18-PRO1. The conditions used for the PCR amplification were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. The PstI-to-KpnI GFP fragment from pMD18-GFP was ligated into PstI-KpnI-digested pRK415 to create pRK415-GFP, and the HindIII-to-PstI EpoPro fragment from pMD18-PRO1 was ligated into HindIII–PstI-digested pRK415-GFP, resulting in pRP-GFP. Conjugation into S. cellulosum So ce90 Ten milliliter batch cultures of E. coli strain S17-1 containing the constructed plasmid pRP-GFP or pRK415-GFP were grown overnight at 37°C in LB medium containing tetracycline. The cultures were used for the inoculation of 100 ml of LB medium, in which the cells were grown at 37°C to an OD600 of 0.6. Then, cells were centrifugated at 4000 rpm for 7 min at room temperature and resuspended to 1010 cells/ml. 100-ml batch cultures of S. cellulosum So ce90 in 250-ml Erlenmeyer flasks were incubated at 30°C in GY medium at 180 rpm for about 6 d, typically resulting in a cell density of approximately 109 cells/ml. After centrifugation at 4000 rpm for 7 min and washing with GY medium at room temperature, the cells were resuspended to 1010 cells/ml. Each suspension (100 µl) were mixed, spotted onto a VY/2 agar plate and incubated at 30°C for 60 h. Then cells from the agar plate were scraped and resuspended in 1 ml of GY medium and 100 µl of the resulting suspension was plated on VY/2 agar containing tetracycline (50 µg/ml) and kanamycin sulfate (10 µg/ml). Colonies were observed after 6–8 d of growth at 30°C in the dark. Transformants were inoculated into fresh VY/2 medium containing tetracycline and kanamycin sulfate. Fluorescence assays To detect fluorescence in transformed S. cellulosum, transformation colonies were incubated for 4 to 5 d at 30°C on VY/2 agar containing tetracycline (50 µg/ml) and kanamycin sulfate (10 µg/ml). Cells were suspended in a drop of formalin on a microscope slide and examined under a BX51 fluorescence microscope (Olympus, Tokyo). Fluorescence images were obtained at an excitation wavelength range of 460–490 nm with emission at 520 nm.
TABLE 1. Strains and plasmids Strain Relevant characteristics Source/Reference Sorangium cellulosum DSMZ So ce90 Wild type; Kanr; DSM 6773 This work S. cellulosum/pRP-GFP Kanr; Tcr; GFP expression; This work S. cellulosum/pRK415-GFP Kanr; Tcr; no GFP expression; E. coli JM109 pir+ cloning strain BioDev S17-1 λ pir+ mating strain Inst Plant Prot. CAAS Plasmids Takara pMD18-T Ampr; lacZ; cloning vector pGFP2.1 Contains GFP cassette Constructed by our lab 17 pBBR1MCS-3 Tcr; broad host range 16 pRK415 Tcr; pRK404 of RK2 family derivative This work pRK415-GFP Tcr; contains promoterless gfp pMD18-Pro 0.89-kb fragment from So ce90 This work This work pRP-GFP Tcr; contains 0.89-kb fragment from So ce90 and promoterless gfp r r Amp , Ampicillin resistance; Kan , kanamycin resistance; Tcr, tetracycline resistance; GFP, green fluorescent protein; DSMZ, German Collection of Microorganisms and Cell Cultures; BioDev-Tech. Scientific & Technical Co., Ltd., China; Inst Plant Prot. CAAS, Institution of Plant Protection, Chinese Academy of Agricultural Sciences, China.
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Southern hybridization A tetracycline fragment (318 bp) probes were generated by PCR amplification from the plasmid pRK415 with the forward primer TetA-F (GCCAATCTTGCTCGT CTCG) and reverse primer TetA-R (GAAACAGCCCGTAGGAA AT) in a PCR reaction. The conditions used for the PCR amplification were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. GFP probes were generated according to the above-mentioned method of constructing the expression plasmid pRP-GFP. The probes used for Southern hybridizations were labeled with [α-32P]dCTP using the Prime-aGene Labeling system (Promega, Madison, WI, USA). Hybridization was performed according to the standard procedures (14). Genomic DNA and the plasmid pRP-GFP were digested using appropriate enzymes, and then were blotted onto a positively charged nylon membrane. Apart from these, uncut genomic DNA and the plasmid pRP-GFP were also analyzed by hybridization using GFP probes. The membrane was UV-cross-linked using a Stratalinker (Stratagene, La Jolla, CA, USA) and then dried for 2 h at 80°C. After 12 h of prehybridization in formamide buffer (5 ×SSPE, 50% formamide, 0.5% SDS, 5 ×Denhardt’s, 100 µg/ml denaturated salmon sperm DNA) at 42°C, the denaturated probes were added to 10 ml of formamide buffer. The hybridization lasted for 24 h at 42°C. After stringent washes, the membrane was exposed to X-ray film inside a cassette between two intensifying screens for 12 h at −70°C to obtain an autoradiography image.
RESULTS Development of antibiotic selection system for S. cellulosum transformants The antibiotic sensitivity of S. cellulosum So ce90 was evaluated to develop a selection system for transformants. In fact, all the Sorangium strains analyzed are resistant to kanamycin (10). We confirmed that the strains could grow on VY/2 medium containing 100 µg/ml kanamycin, but they could not be found on the plates containing 40 µg/ml tetracycline. On the basis of these results, a simple and effective selection system was developed, by which S. cellulosum transformants were selected on media containing kanamycin and tetracycline after conjugation, while kanamycin was used to inhibit the E. coli donor strains. Construction and transformation of plasmid pRP-GFP Figure 1 shows the construction procedure for the expression plasmid pRP-GFP (about 12 kb). EpoA encodes EPOS A (1421 amino acids), a type I modular of a polyketide synthase (PKS) which is presumed to be involved in the initiation of epothilone biosynthesis through the loading of an acetate unit onto the multienzyme complex (13, 15). The promoterless GFP-coding sequence is located immediately after the EpoPro fragment. The conditions that might induce an increase in the transfer frequency were investigated. Firstly, before mating with the donor E. coli, S. cellulosum So ce90 was heated in a water bath at different temperatures for different times as described by Jaoua et al. (9). The results showed that the transfer frequency did not change by heating the S. cellulosum cells after transconjugation. Secondly, the mating time was changed from 35 to 80 h, and the results indicated that the mating time was more important for transfer frequency, of which the highest frequency was observed in the range of 60–65 h. The frequency of transfer to S. cellulosum So ce90
FIG. 1. Construction procedure for plasmid pRP-GFP. pRP-GFP contains 890 bp of the EpoPro fragment, which lie upstream of the epoA of So ce90. The gfp coding sequence is located downstream of EpoPro. The plasmid also contains a tetracycline resistance cassette. The unique restriction enzymes sites are indicated.
(calculated per viable Sorangium cell) was in the range 5– 9 ×10–6. However, when the mating time was less than 40 h or more than 70 h, few transformants were found. Expression of plasmid pRP-GFP in S. cellulosum So ce90 The S. cellulosum So ce90 containing the plasmid pRP-GFP exhibited a green light (Fig. 2) under fluorescence microscopy after conjugation. In contrast, no fluorescence was detected in the wild-type So ce90 or the recombinant E. coli. To investigate the potential function of the fragment of EpoPro in S. cellulosum So ce90, the plasmid pRK415-GFP, which lacked the EpoPro fragment, was also transformed into the bacteria as a control in our experiment. The result showed that none of the recombinants containing the plasmid pRK415-GFP exhibited fluorescence, which indicates that the expression of gfp in the pRP-GFP transformants was driven by the EpoPro. This phenomenon hinted that EpoPro is a promoter-activity DNA fragment within the plasmid pRPGFP. Stability of transformants The stability of the S. cellulosum So ce90 transformants containing the plasmid pRPGFP was tested. Single transformant colonies were streaked on tetracycline and kanamycin VY/2 agar and incubated. Single colonies were selected and inoculated into 5 ml of G52 medium with or without antibiotics. After 4 d of incubation, which corresponds to 6 generation times of S. cellulosum So ce90, 100 µl of the suspensions was inoculated into 5 ml of fresh G52 medium with or without antibiotics. This was repeated seven times. The cells for 50 generations obtained
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FIG. 2. (A) Fluorescence micrograph of S. cellulosum So ce90 containing plasmid pRP-GFP. The green color in the S. cellulosum So ce90 is due to GFP. (B) Phase-contrast image of the same sample. (C) Wildtype S. cellulosum So ce90 did not fluoresce significantly. (D) Phasecontrast image of wild-type S. cellulosum So ce90.
from the procedure above were counted and then visualized under a fluorescence microscope. The percentage of fluorescence cells was determined and taken as a measure of transformant stability. All cells retained fluorescence in the case of the antibiotic incubation, whereas 73% of the cells retained fluorescence in the case of the antibiotic-free incubation. The fluorescence found in the subculture indicated that the plasmid was stably maintained in the bacteria with selective pressure. Molecular analysis of DNA of transformants of S. cellulosum So ce90 Free plasmid could not be found when the total DNA of the transformants was extracted and electrophoresed on an in agarose gel, which suggested that the plasmids in the transformants were either maintained at a relatively low copy number or were integrated into the chromosome of the host strain. To investigate the characteristics of the plasmid pRP-GFP in S. cellulosum more clearly, we analyzed the transformants in detail by carrying out several restriction enzyme digestions of DNA and Southern blot hybridization with a 726-bp GFP fragment and a 318-bp tetracycline fragment as the probe, respectively. According to the restriction map (Fig. 1) for the selected restriction enzymes, there was a single site for StuI, EcoRI, HindIII and KpnI within pRP-GFP, whereas there was no site for XbaI. The plasmid pRP-GFP, S. cellulosum/pRP-GFP DNA and S. cellulosum So ce90 DNA were isolated and digested completely with StuI, EcoRI-HindIII, KpnI, and KpnI-XbaI and hybridized with GFP or tetracycline fragments as the probe, respectively. Upon StuI and EcoRI-HindIII digestion with the GFP fragment as a probe, pRP-GFP and S. cellulosum/pRP-GFP DNA exhibited the same bands of 12.0 kb and 1.6 kb respectively, while wild-type S. cellulosum So ce90 DNA exhibited no band at all (Fig. 3). When digested by KpnI and KpnI-XbaI with the tetracycline fragment as a probe, pRP-GFP and S. cellulosum/pRP-GFP DNA exhibited the same band of 12.0 kb, respectively, while wild-type S. cellulosum So ce90 DNA exhibited no band at all (Fig. 4). Moreover, uncut pRP-GFP, S. cellulosum/pRP-GFP DNA and S. cellulosum So ce90 DNA were also extracted and hybridized with GFP fragments as the probe. The results show no difference in the hybridization patterns between uncut pRP-GFP and S. cellulosum/pRP-GFP DNA, while no hybridization signal occurred with uncut So ce90 DNA (Fig. 5).
FIG. 3. Southern hybridization analysis of S. cellulosum/pRP-GFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with GFP from plasmid pRP-GFP. Lane 1, StuI-digested S. cellulosum/pRP-GFP DNA; lane 2, StuI-digested wild-type strain So ce90 DNA; lane 3, (EcoRI-HindIII)-digested S. cellulosum/pRP-GFP DNA; lane 4, (EcoRI-HindIII)-digested wild-type strain So ce90 DNA; lane 5, StuI-digested plasmid pRP-GFP; lane 6, (EcoRI-HindIII)-digested plasmid pRP-GFP.
FIG. 4. Southern hybridization analysis of S. cellulosum/pRP-GFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with tetracycline fragment from plasmid pRP-GFP. Lane 1, KpnI-digested plasmid pRP-GFP; lane 2, KpnI-digested S. cellulosum/pRP-GFP DNA; lane 3, KpnI-digested wild-type strain So ce90 DNA; lane 4, (KpnI-XbaI)-digested plasmid pRP-GFP; lane 5, (KpnI-XbaI)-digested S. cellulosum/pRP-GFP DNA; lane 6, (KpnI-XbaI)-digested wild-type strain So ce90 DNA.
Taken together, all the results thereafter strongly suggested that the plasmid pRP-GFP could be replicated autonomously in S. cellulosum without being integrated into the chromosome. DISCUSSION In this study, the plasmid pRP-GFP derived from the plasmid pRK415 was successfully constructed for transfer from E. coli to S. cellulosum and could be employed as a tool for the manipulation of gene expression in S. cellulosum. This
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FIG. 5. Southern hybridization analysis of uncut S. cellulosum/pRPGFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with GFP from plasmid pRP-GFP. Lane 1, S. cellulosum/pRP-GFP DNA; lane 2, wild-type strain So ce90 DNA; lane 3, plasmid pRPGFP.
appears to be the first exogenous plasmid that can be stably maintained as an extrachromosomal element in S. cellulosum. The plasmid pRK415 is a tetracycline-resistant (Tet (A)) broad-host-range vector that is a self-transmissive IncP plasmid. The plasmid is more versatile for use in many gramnegative hosts other than E. coli such as in the Rhodobacter species of photosynthetic bacteria (16). As far as the low copy number of pRK415 in hosts was concerned, the plasmid pBBR1MCS, another broad-host-range vector with several antibiotic resistance genes (17, 18), which was derived from Bordatella brochiseptica, can be maintained at a medium copy number in hosts such as Brucella (19), Vibrio, Escherichia, Pseudomonas and Rhizobium (20). Thus, given its higher copy number and smaller size (between 4.7 and 5.3 kb), pBBR1MCS might be more useful for developing expression vectors. In the first series of experiments we investigated the transfer of pRK415 and pBBR1MCS-3 from E. coli S17-1 to S. cellulosum So ce90 by conjugation. The results indicated that the transfer of the plasmid pRK415 could be detected when selected using kanamycin and tetracycline; unfortunately, no tranformants of pBBR1MCS-3 could be found. On the basis of the above experiment result, we selected pRK415 as an initial plasmid to construct the pRP-GFP vector. The results of all the Southern blots analyses (Figs. 3 and 4) showed no difference in the restriction pattern between the plasmid pRP-GFP and S. cellulosum/pRP-GFP DNA, which confirmed that the plasmid could be replicated autonomously without being integrated into the chromosome of S. cellulosum So ce90. Additional evidence could further support for our conclusion on the basis of the analysis and in addition to a comparison of the restriction enzyme site with the 68,750-bp partially known So ce90 genomic sequence (accession no. AF210843) accessed from GenBank. Within the So ce90 genomic sequence involved in the synthesis of epothilones, there are 14 digestion sites for StuI, 2 sites for EcoRI, 6 sites for HindIII, 9 sites for KpnI and 1 site for XbaI. Thus, the Southern blot results could hardly be of an integrated plas-
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mid that happens to have the same enzyme sites exactly positioned to give the same size as a replicating one after being digested by several restriction enzymes. Moreover, the same hybridization patterns between uncut pRP-GFP and S. cellulosum/pRP-GFP DNA showed that the undigested DNA gave the same mobility band corresponding to the covalently closed circular plasmid. The results strongly confirmed the proposal that the introduced DNA was maintained as a plasmid in S. cellulosum So ce90. However, the plasmid pRP-GFP in the So ce90 transformants could not be detected using standard ethidium bromide staining, although a large amount of the plasmid pRP-GFP could be extracted from E. coli. This result suggested that the copy number became lower in the host strain. Low-copy-number plasmid vectors are sometimes useful for the cloning of a foreign gene, particularly when it codes for a toxic substance. So far, it has been difficult and complicated to introduce foreign DNA into S. cellulosum by conjugation. This bacterium is naturally multiresistant against most commonly used antibiotics and often, genetic methods established for one strain cannot be applied to others, even if they are phylogenetically closely related (12). Using biparental and triparental mating, Kopp et al. (11) has developed methodologies for DNA transfer from E. coli via conjugation to the strain So ce56, but they found that the same conjugation protocol was not applicable to So ce12. Thus, although pRPGFP had successfully transconjugated into So ce90 in our experiment, it was not unknown whether pRP-GFP could be used for to other S. cellulosum strains. Further research is needed to confirm this. Moreover, a worthwhile investigation concerned the transconjugant frequency of the system of gene transfer to S. cellulosum. In the first system, which was established by Jaoua et al. (9) with the vector pSUP2021 derived from pBR325, a plasmid with a Tn5 transposon, it was found that integrating DNA with regions of homology of less than 1000 bp could be extremely difficult because the recombination frequency of the host is very low for homology recombination. Obviously, without the occurrence of homology recombination, the transfer might be operable using the autonomously replicating plasmid pRP-GFP with smaller fragments. Furthermore, some attempts have been made to increase the transfer frequency using heat shock. Jaoua et al. (9) raised the frequency by a factor 10 by heating S. cellulosum cells at 50°C for 10 min. However, we found that the frequency of transposition did not change using the same manipulation technique, which is consistent with the result of Julien and Fehd (8). We found that mating time was closely related to the frequency. To obtain the highest frequency, it is necessary to avoid excessively long or short mating time. In this study, the GFP reporter system in S. cellulosum was also first established. GFP is the most recent and most popular reporter, since it has numerous advantages over other systems, being stable to temperature, pH, and denaturing agents. It is easily and nondestructively detected by illumination with blue or near-UV light and, unlike all other systems, requires no substrate. Furthermore, because the fluorescence of GFP is linearly proportional to the amount of the protein, it is an attractive reporter for monitoring in situ
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gene expression (21). S. cellulosum is difficult to engineer owing to the low efficiency of introducing DNA into it and the limited numbers of molecular tools and markers that have been developed for the organism (8, 9, 11). The stable expression of GFP in S. cellulosum was rapid and convenient for the conjugation screening of the transformants of the bacterium. Although the gene cluster responsible for the biosynthesis of epothilones in S. cellulosum So ce90 has been cloned and sequenced, the promoter of this operon is still unknown. Molnár et al. (13) considered that the promoter might lie in the stretch immediately preceding epoA in a 935-bp noncoding region. In our experiments, the comparison of the expression of gfp within pRP-GFP with that within pRK415GFP in S. cellulosum confirmed that the EpoPro fragment had the function of a promoter that could drive the expression of the gene in the bacterium. However, noweverHo GFP expression was detected in E. coli, which might be attributable to either the promoter not be recognized by E. coli RNA polymerase or other transcription factors required for the transcription. Further research on the properties of the EpoPro fragment as a promoter would be interesting. To date, no naturally occurring plasmids have been described in myxobacteria and no broad-host-range vectors are able to replicate in myxobacteria. Therefore, the work reported here will provide a powerful tool for isolating and analyzing the genes involved in epothilone metabolism. In this sense, this work may be a breakthrough in the genetics and molecular biology of S. cellulosum So ce90. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 30740474). We thank Shu-Sheng Yang of China Agricultural University for generously providing E. coli strain S17-1. We also thank Xu-Qing Chen of the Beijing Research Center of Agro-Biotechnology for helpful discussions and the critical reading of the manuscript.
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Drugs. Future, 27, 1149–1163 (2002). 6. Hardt, I., Steinmetz, H., Gerth, K., Sasse, F., Reichenbach, H., and Höfle, G.: New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/D13: isolation, structure elucidation, and SAR studies. J. Nat. Prod., 64, 847– 856 (2001). 7. Gerth, K., Pradella, S., Perlova, O., Beyer, S., and Müller, R.: Myxobacteria: proficient producers of novel natural products with various biological activities-past and future biotechnological aspects with the focus on the genus Sorangium. J. Biotechnol., 106, 233–253 (2003). 8. Julien, B. and Fehd, R.: Development of a mariner-based transposon for use in Sorangium cellulosum. Appl. Environ. Microbiol., 69, 6299–6301 (2003). 9. Jaoua, S., Neff, S., and Schupp, T.: Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome. Plasmid, 28, 157–165 (1992). 10. Pradella, S., Hans, A., Spröer, C., Reichenbach, H., Gerth, K., and Beyer, S.: Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56. Arch. Microbiol., 178, 484–492 (2002). 11. Kopp, M., Irschik, H., Gross, F., Perlova, O., Sandmann, A., Gerth, K., and Müller, R.: Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce 12 and So ce56: development of a mariner-based transposon mutagenesis system. J. Biotechnol., 107, 29–40 (2004). 12. Bode, H. B. and Müller, R.: Analysis of myxobacterial secondary metabolism goes molecular. J. Ind. Microbiol. Biotechnol., 33, 577–588 (2006). 13. Molnár, I., Schupp, T., Ono, M., Zirkle, R. E., Milnamow, M., Nowak-Thompson, B., Engel, N., Toupet, C., Stratmann, A., Cry, D. D., Gorlach, J., Mayo, J. M., Hu, A., Goff, S., Schmid, J., and Ligon, J. M.: The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. Chem. Biol., 7, 97–109 (2000). 14. Rapley, R.: The nucleic acid protocols handbook, p. 77–80. Humana Press, New Jersey (2000). 15. Julien, B., Shah, S., Ziermann, R., Goldman, R., Katz, L., and Khosla, C.: Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum. Gene, 249, 153–160 (2000). 16. Lee, J. K., Kiley, P. J., and Kaplan, S.: Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides. J. Bacteriol., 171, 3391–3405 (1989). 17. Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M., II, and Peterson, K. M.: pBBR1MCS: a broad-host-range cloning vector. Biotechniques, 16, 800–802 (1994). 18. Kovach, M. E., Elzer, P. H., Hill, D. S., Roberston, G. T., Farris, M. A., Roop R. M., II, and Peterson, K. M.: Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene, 166, 175–176 (1995). 19. Köhler, S., Ouahrani-Bettache, S., Layssac, M., Teyssier, J., and Liautard, J. P.: Constitutive and inducible expression of green fluorescent protein in Brucella suis. Infect. Immun., 67, 6695–6697 (1999). 20. Antoine, R. and Locht, C.: Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from grampositive organisms. Mol. Microbiol., 6, 1785–1799 (1992). 21. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C.: Green fluorescent protein as a marker for gene expression. Science, 263, 802–805 (1994).
© 2007, The Society for Biotechnology, Japan
Autonomously Replicating Plasmid Transforms Sorangium cellulosum So ce90 and Induces Expression of Green Fluorescent Protein Yun Tu,1 Guo-Ping Chen,1 and Yuan-Liang Wang1* College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China1 Received 30 March 2007/Accepted 3 August 2007
A recombinant plasmid, pRP-GFP, harboring the green fluorescent protein (GFP) gene from the broad-host-range mobilizable plasmid pRK415 of the RK2 family was constructed and transferred by conjugation from Escherichia coli S17-1 to Sorangium cellulosum So ce90. The results of Southern blot analysis showed that the plasmid replicated autonomously without being integrated into the chromosome of the bacterium. In pRP-GFP, gfp was driven by a So ce90 DNA fragment called EpoPro, which is an 890-bp DNA segment spanning from −890 to −1 relative to the start codon (GTG) of epoA, a gene that encodes EPOS A, which is presumed to be involved in the initiation of epothilone biosynthesis. The GFP in the transformed S. cellulosum So ce90 was detected by fluorescence microscopy, which suggests that the EpoPro fragment had the function of promoter. Because the green fluorescent bacilli could be directly observed by fluorescence microscopy, the stable expression of GFP was rapid and convenient for conjugation screening in addition to the antibiotic resistance genes within the constructed plasmid. This is the first report on an exogenous plasmid that can be stably maintained as an extrachromosomal element in S. cellulosum. [Key words: Sorangium cellulosum, autonomously replicating plasmid, green fluorescent protein, conjugation, epothilone]
into the chromosome of the host in such transfer systems. Nevertheless, for homology recombination, integrating DNA with regions of homology of less than 1000 bp could be extremely difficult because the recombination frequency in the host is very low (9). Julien and Fehd (8) showed that the transposition of a transposon into So ce90 is rare (1 in 10,000 to 1 in 100,000). So far, no endogenous plasmid had been found in S. cellulosum, and few attempts to transfer an autonomously replicating exogenous plasmid into this species have been reported (12). Such plasmid would be of great value for genetic research using bacteria. In this paper, we constructed a plasmid, pRP-GFP, that can be transferred to So ce90 easily and replicated autonomously without being integrated into the chromosome of S. cellulosum. Our results indicate that the plasmid is a fastacting, convenient and valuable genetic tool for research on So ce90. Furthermore, epothilone biosynthetic gene clusters are made up of seven genes transcribed in the same direction, encoding five polyketide synthases (PKSs) (epoA-epoE), a nonribosomal peptide synthetase (NRPS) (epoP) and a cytochrome P450 (epoF) (13). Although the gene cluster has been sequenced, the promoter of this operon is still unknown. In this study, we found that the fragment EpoPro, which was used to construct pRP-GFP, spanning from −890 to −1 relative to the start codon (GTG) of epoA, could drive the expression of gfp in S. cellulosum. It is suggested that this fragment has the function of a promoter.
The gram-negative bacterium Sorangium cellulosum belongs to the order Myxococcales, which is composed of soildwelling bacteria with complex social behaviors and multicellular developmental processes (1). Over the past 20 years, S. cellulosum have become well known for their secondary metabolites with unique structures and biological activities, e.g., epothilones (2), soraphens (3), disorazols, and chivosazoles (4). Of these bioactive products, epothilones have attracted considerable attention as a new class of active microtubule-stabilising cytotoxic agents for the treatment of solid tumors by means of the same mechanism of action as that of Taxol (2). Above all, epothilones have more advantages than Taxol, such as better water solubility, simple structures and the activity of the P-glycoprotein (5). The marked potential for epothilones makes Sorangium species very interesting test subjects for molecular biological studies of genetic control and molecular mechanisms. Of the series of Sorangium strains that have been isolated, So ce90 has received the most attention because this strain was shown to excrete more than 30 different epothilones (6). However, Sorangium strains are some of the most difficult myxobacteria to work with. They have the longest doubling time of any myxobacterium, up to 16 h, and very few genetic tools are available (7, 8). Even now, conjugation in S. cellulosum has been established to introduce DNA into the bacterium through homology recombination or transposons (8–11). All the results show that foreign DNA is integrated * Corresponding author. e-mail: [email protected] phone/fax: +86-23-65102508 385
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MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions The strains and plasmids used in this study are listed in Table 1. A seed culture of S. cellulosum So ce90 (DSM 6773) was obtained from the German Collection of Microorganisms and Cell Cultures. It was grown at 30°C in liquid G52 medium and plated on VY/2 agar plates as described by Gerth et al. (2). GY medium was used for conjugation. It contained 2 g/l glucose, 2 g/l yeast extract, 8 mg/l Na-FeIII-EDTA, 1 g/l MgSO4 ⋅7H2O, 1 g/l CaCl2 ⋅2H2O and 11.5 g/l HEPES. The medium was adjusted to pH 7.4 with KOH and autoclaved for 30 min at 121°C. If required, the media were supplemented with 50 µg/ml tetracycline (Sigma, St. Louis, MO, USA) and 10 µg/ml kanamycin sulfate (Sigma). Escherichia coli strains JM109 and S17-1 were used for plasmid subcloning and conjugation, respectively. E. coli strains were grown in LB medium at 37°C. When necessary, antibiotics were used at the following concentrations: 50 µg/ml ampicillin (Sigma), 50 µg/ml kanamycin sulfate, and 50 µg/ml tetracycline. E. coli S17-1 with a plasmid was used as a conjugation donor. DNA preparation S. cellulosum So ce90 genomic DNA was isolated using the Genomic DNA Purification kit (BioDev, Beijing, China) according to the manufacturer’s protocol. Plasmid DNA purification was carried out using E.Z.N.A Plasmid Miniprep kit I (Omega, Doraville, GA, USA) according to the manufacturer’s protocol. Construction of expression plasmid pRP-GFP All the restriction enzymes used, Taq polymerase and T4 DNA ligase were purchased from Takara (Dalian, Liaoning, China) and used according to the manufacturer’s recommendations. The PCR products were directly ligated into pMD18-T using the pMD18-T vector kit (Takara) according to the manufacture’s protocol and sequenced by Invitrogen Biotechnology (Shanghai, China). The pMD18-T vector, a specific TA cloning plasmid for cloning of PCR products, was rebuilt from pUC18 by Takara company. The promoterless GFP DNA segment was amplified from the plasmid pGFP2.1, using the forward primer containing a PstI site and the ATG initiation codon (GFP-F: 5′-CTGCAGATGGGTAGT AAAGGAGAAG-3′) and the reverse primer containing a KpnI site (GFP-R: 5′-GGTACCCTATTTGTATAGTTCATCC-3′). The conditions used for the PCR reaction were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. The 726-bp GFP fragment was ligated into the plasmid pMD18-T to
create pMD18-GFP. The 890-bp EpoPro fragment, spanning from −890 to −1 relative to the start codon (GTG) of epoA was PCR amplified from S. cellulosum So ce90 DNA with the forward primer containing a HindIII site (Pro1F: 5′-AAGCTTCCTGCCTCGATGGCCTTCCC3′) and the reverse primer containing a PstI site (Pro1R: 5′-CTGC AGGACGGGCACATCCTCAGCG-3′), and then ligated into the plasmid pMD18-T to create pMD18-PRO1. The conditions used for the PCR amplification were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. The PstI-to-KpnI GFP fragment from pMD18-GFP was ligated into PstI-KpnI-digested pRK415 to create pRK415-GFP, and the HindIII-to-PstI EpoPro fragment from pMD18-PRO1 was ligated into HindIII–PstI-digested pRK415-GFP, resulting in pRP-GFP. Conjugation into S. cellulosum So ce90 Ten milliliter batch cultures of E. coli strain S17-1 containing the constructed plasmid pRP-GFP or pRK415-GFP were grown overnight at 37°C in LB medium containing tetracycline. The cultures were used for the inoculation of 100 ml of LB medium, in which the cells were grown at 37°C to an OD600 of 0.6. Then, cells were centrifugated at 4000 rpm for 7 min at room temperature and resuspended to 1010 cells/ml. 100-ml batch cultures of S. cellulosum So ce90 in 250-ml Erlenmeyer flasks were incubated at 30°C in GY medium at 180 rpm for about 6 d, typically resulting in a cell density of approximately 109 cells/ml. After centrifugation at 4000 rpm for 7 min and washing with GY medium at room temperature, the cells were resuspended to 1010 cells/ml. Each suspension (100 µl) were mixed, spotted onto a VY/2 agar plate and incubated at 30°C for 60 h. Then cells from the agar plate were scraped and resuspended in 1 ml of GY medium and 100 µl of the resulting suspension was plated on VY/2 agar containing tetracycline (50 µg/ml) and kanamycin sulfate (10 µg/ml). Colonies were observed after 6–8 d of growth at 30°C in the dark. Transformants were inoculated into fresh VY/2 medium containing tetracycline and kanamycin sulfate. Fluorescence assays To detect fluorescence in transformed S. cellulosum, transformation colonies were incubated for 4 to 5 d at 30°C on VY/2 agar containing tetracycline (50 µg/ml) and kanamycin sulfate (10 µg/ml). Cells were suspended in a drop of formalin on a microscope slide and examined under a BX51 fluorescence microscope (Olympus, Tokyo). Fluorescence images were obtained at an excitation wavelength range of 460–490 nm with emission at 520 nm.
TABLE 1. Strains and plasmids Strain Relevant characteristics Source/Reference Sorangium cellulosum DSMZ So ce90 Wild type; Kanr; DSM 6773 This work S. cellulosum/pRP-GFP Kanr; Tcr; GFP expression; This work S. cellulosum/pRK415-GFP Kanr; Tcr; no GFP expression; E. coli JM109 pir+ cloning strain BioDev S17-1 λ pir+ mating strain Inst Plant Prot. CAAS Plasmids Takara pMD18-T Ampr; lacZ; cloning vector pGFP2.1 Contains GFP cassette Constructed by our lab 17 pBBR1MCS-3 Tcr; broad host range 16 pRK415 Tcr; pRK404 of RK2 family derivative This work pRK415-GFP Tcr; contains promoterless gfp pMD18-Pro 0.89-kb fragment from So ce90 This work This work pRP-GFP Tcr; contains 0.89-kb fragment from So ce90 and promoterless gfp r r Amp , Ampicillin resistance; Kan , kanamycin resistance; Tcr, tetracycline resistance; GFP, green fluorescent protein; DSMZ, German Collection of Microorganisms and Cell Cultures; BioDev-Tech. Scientific & Technical Co., Ltd., China; Inst Plant Prot. CAAS, Institution of Plant Protection, Chinese Academy of Agricultural Sciences, China.
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Southern hybridization A tetracycline fragment (318 bp) probes were generated by PCR amplification from the plasmid pRK415 with the forward primer TetA-F (GCCAATCTTGCTCGT CTCG) and reverse primer TetA-R (GAAACAGCCCGTAGGAA AT) in a PCR reaction. The conditions used for the PCR amplification were as follows: initial denaturation for 3 min at 94°C, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, and a final 10-min extension at 72°C. GFP probes were generated according to the above-mentioned method of constructing the expression plasmid pRP-GFP. The probes used for Southern hybridizations were labeled with [α-32P]dCTP using the Prime-aGene Labeling system (Promega, Madison, WI, USA). Hybridization was performed according to the standard procedures (14). Genomic DNA and the plasmid pRP-GFP were digested using appropriate enzymes, and then were blotted onto a positively charged nylon membrane. Apart from these, uncut genomic DNA and the plasmid pRP-GFP were also analyzed by hybridization using GFP probes. The membrane was UV-cross-linked using a Stratalinker (Stratagene, La Jolla, CA, USA) and then dried for 2 h at 80°C. After 12 h of prehybridization in formamide buffer (5 ×SSPE, 50% formamide, 0.5% SDS, 5 ×Denhardt’s, 100 µg/ml denaturated salmon sperm DNA) at 42°C, the denaturated probes were added to 10 ml of formamide buffer. The hybridization lasted for 24 h at 42°C. After stringent washes, the membrane was exposed to X-ray film inside a cassette between two intensifying screens for 12 h at −70°C to obtain an autoradiography image.
RESULTS Development of antibiotic selection system for S. cellulosum transformants The antibiotic sensitivity of S. cellulosum So ce90 was evaluated to develop a selection system for transformants. In fact, all the Sorangium strains analyzed are resistant to kanamycin (10). We confirmed that the strains could grow on VY/2 medium containing 100 µg/ml kanamycin, but they could not be found on the plates containing 40 µg/ml tetracycline. On the basis of these results, a simple and effective selection system was developed, by which S. cellulosum transformants were selected on media containing kanamycin and tetracycline after conjugation, while kanamycin was used to inhibit the E. coli donor strains. Construction and transformation of plasmid pRP-GFP Figure 1 shows the construction procedure for the expression plasmid pRP-GFP (about 12 kb). EpoA encodes EPOS A (1421 amino acids), a type I modular of a polyketide synthase (PKS) which is presumed to be involved in the initiation of epothilone biosynthesis through the loading of an acetate unit onto the multienzyme complex (13, 15). The promoterless GFP-coding sequence is located immediately after the EpoPro fragment. The conditions that might induce an increase in the transfer frequency were investigated. Firstly, before mating with the donor E. coli, S. cellulosum So ce90 was heated in a water bath at different temperatures for different times as described by Jaoua et al. (9). The results showed that the transfer frequency did not change by heating the S. cellulosum cells after transconjugation. Secondly, the mating time was changed from 35 to 80 h, and the results indicated that the mating time was more important for transfer frequency, of which the highest frequency was observed in the range of 60–65 h. The frequency of transfer to S. cellulosum So ce90
FIG. 1. Construction procedure for plasmid pRP-GFP. pRP-GFP contains 890 bp of the EpoPro fragment, which lie upstream of the epoA of So ce90. The gfp coding sequence is located downstream of EpoPro. The plasmid also contains a tetracycline resistance cassette. The unique restriction enzymes sites are indicated.
(calculated per viable Sorangium cell) was in the range 5– 9 ×10–6. However, when the mating time was less than 40 h or more than 70 h, few transformants were found. Expression of plasmid pRP-GFP in S. cellulosum So ce90 The S. cellulosum So ce90 containing the plasmid pRP-GFP exhibited a green light (Fig. 2) under fluorescence microscopy after conjugation. In contrast, no fluorescence was detected in the wild-type So ce90 or the recombinant E. coli. To investigate the potential function of the fragment of EpoPro in S. cellulosum So ce90, the plasmid pRK415-GFP, which lacked the EpoPro fragment, was also transformed into the bacteria as a control in our experiment. The result showed that none of the recombinants containing the plasmid pRK415-GFP exhibited fluorescence, which indicates that the expression of gfp in the pRP-GFP transformants was driven by the EpoPro. This phenomenon hinted that EpoPro is a promoter-activity DNA fragment within the plasmid pRPGFP. Stability of transformants The stability of the S. cellulosum So ce90 transformants containing the plasmid pRPGFP was tested. Single transformant colonies were streaked on tetracycline and kanamycin VY/2 agar and incubated. Single colonies were selected and inoculated into 5 ml of G52 medium with or without antibiotics. After 4 d of incubation, which corresponds to 6 generation times of S. cellulosum So ce90, 100 µl of the suspensions was inoculated into 5 ml of fresh G52 medium with or without antibiotics. This was repeated seven times. The cells for 50 generations obtained
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FIG. 2. (A) Fluorescence micrograph of S. cellulosum So ce90 containing plasmid pRP-GFP. The green color in the S. cellulosum So ce90 is due to GFP. (B) Phase-contrast image of the same sample. (C) Wildtype S. cellulosum So ce90 did not fluoresce significantly. (D) Phasecontrast image of wild-type S. cellulosum So ce90.
from the procedure above were counted and then visualized under a fluorescence microscope. The percentage of fluorescence cells was determined and taken as a measure of transformant stability. All cells retained fluorescence in the case of the antibiotic incubation, whereas 73% of the cells retained fluorescence in the case of the antibiotic-free incubation. The fluorescence found in the subculture indicated that the plasmid was stably maintained in the bacteria with selective pressure. Molecular analysis of DNA of transformants of S. cellulosum So ce90 Free plasmid could not be found when the total DNA of the transformants was extracted and electrophoresed on an in agarose gel, which suggested that the plasmids in the transformants were either maintained at a relatively low copy number or were integrated into the chromosome of the host strain. To investigate the characteristics of the plasmid pRP-GFP in S. cellulosum more clearly, we analyzed the transformants in detail by carrying out several restriction enzyme digestions of DNA and Southern blot hybridization with a 726-bp GFP fragment and a 318-bp tetracycline fragment as the probe, respectively. According to the restriction map (Fig. 1) for the selected restriction enzymes, there was a single site for StuI, EcoRI, HindIII and KpnI within pRP-GFP, whereas there was no site for XbaI. The plasmid pRP-GFP, S. cellulosum/pRP-GFP DNA and S. cellulosum So ce90 DNA were isolated and digested completely with StuI, EcoRI-HindIII, KpnI, and KpnI-XbaI and hybridized with GFP or tetracycline fragments as the probe, respectively. Upon StuI and EcoRI-HindIII digestion with the GFP fragment as a probe, pRP-GFP and S. cellulosum/pRP-GFP DNA exhibited the same bands of 12.0 kb and 1.6 kb respectively, while wild-type S. cellulosum So ce90 DNA exhibited no band at all (Fig. 3). When digested by KpnI and KpnI-XbaI with the tetracycline fragment as a probe, pRP-GFP and S. cellulosum/pRP-GFP DNA exhibited the same band of 12.0 kb, respectively, while wild-type S. cellulosum So ce90 DNA exhibited no band at all (Fig. 4). Moreover, uncut pRP-GFP, S. cellulosum/pRP-GFP DNA and S. cellulosum So ce90 DNA were also extracted and hybridized with GFP fragments as the probe. The results show no difference in the hybridization patterns between uncut pRP-GFP and S. cellulosum/pRP-GFP DNA, while no hybridization signal occurred with uncut So ce90 DNA (Fig. 5).
FIG. 3. Southern hybridization analysis of S. cellulosum/pRP-GFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with GFP from plasmid pRP-GFP. Lane 1, StuI-digested S. cellulosum/pRP-GFP DNA; lane 2, StuI-digested wild-type strain So ce90 DNA; lane 3, (EcoRI-HindIII)-digested S. cellulosum/pRP-GFP DNA; lane 4, (EcoRI-HindIII)-digested wild-type strain So ce90 DNA; lane 5, StuI-digested plasmid pRP-GFP; lane 6, (EcoRI-HindIII)-digested plasmid pRP-GFP.
FIG. 4. Southern hybridization analysis of S. cellulosum/pRP-GFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with tetracycline fragment from plasmid pRP-GFP. Lane 1, KpnI-digested plasmid pRP-GFP; lane 2, KpnI-digested S. cellulosum/pRP-GFP DNA; lane 3, KpnI-digested wild-type strain So ce90 DNA; lane 4, (KpnI-XbaI)-digested plasmid pRP-GFP; lane 5, (KpnI-XbaI)-digested S. cellulosum/pRP-GFP DNA; lane 6, (KpnI-XbaI)-digested wild-type strain So ce90 DNA.
Taken together, all the results thereafter strongly suggested that the plasmid pRP-GFP could be replicated autonomously in S. cellulosum without being integrated into the chromosome. DISCUSSION In this study, the plasmid pRP-GFP derived from the plasmid pRK415 was successfully constructed for transfer from E. coli to S. cellulosum and could be employed as a tool for the manipulation of gene expression in S. cellulosum. This
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FIG. 5. Southern hybridization analysis of uncut S. cellulosum/pRPGFP DNA, wild-type strain So ce90 DNA and plasmid pRP-GFP, probed with GFP from plasmid pRP-GFP. Lane 1, S. cellulosum/pRP-GFP DNA; lane 2, wild-type strain So ce90 DNA; lane 3, plasmid pRPGFP.
appears to be the first exogenous plasmid that can be stably maintained as an extrachromosomal element in S. cellulosum. The plasmid pRK415 is a tetracycline-resistant (Tet (A)) broad-host-range vector that is a self-transmissive IncP plasmid. The plasmid is more versatile for use in many gramnegative hosts other than E. coli such as in the Rhodobacter species of photosynthetic bacteria (16). As far as the low copy number of pRK415 in hosts was concerned, the plasmid pBBR1MCS, another broad-host-range vector with several antibiotic resistance genes (17, 18), which was derived from Bordatella brochiseptica, can be maintained at a medium copy number in hosts such as Brucella (19), Vibrio, Escherichia, Pseudomonas and Rhizobium (20). Thus, given its higher copy number and smaller size (between 4.7 and 5.3 kb), pBBR1MCS might be more useful for developing expression vectors. In the first series of experiments we investigated the transfer of pRK415 and pBBR1MCS-3 from E. coli S17-1 to S. cellulosum So ce90 by conjugation. The results indicated that the transfer of the plasmid pRK415 could be detected when selected using kanamycin and tetracycline; unfortunately, no tranformants of pBBR1MCS-3 could be found. On the basis of the above experiment result, we selected pRK415 as an initial plasmid to construct the pRP-GFP vector. The results of all the Southern blots analyses (Figs. 3 and 4) showed no difference in the restriction pattern between the plasmid pRP-GFP and S. cellulosum/pRP-GFP DNA, which confirmed that the plasmid could be replicated autonomously without being integrated into the chromosome of S. cellulosum So ce90. Additional evidence could further support for our conclusion on the basis of the analysis and in addition to a comparison of the restriction enzyme site with the 68,750-bp partially known So ce90 genomic sequence (accession no. AF210843) accessed from GenBank. Within the So ce90 genomic sequence involved in the synthesis of epothilones, there are 14 digestion sites for StuI, 2 sites for EcoRI, 6 sites for HindIII, 9 sites for KpnI and 1 site for XbaI. Thus, the Southern blot results could hardly be of an integrated plas-
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mid that happens to have the same enzyme sites exactly positioned to give the same size as a replicating one after being digested by several restriction enzymes. Moreover, the same hybridization patterns between uncut pRP-GFP and S. cellulosum/pRP-GFP DNA showed that the undigested DNA gave the same mobility band corresponding to the covalently closed circular plasmid. The results strongly confirmed the proposal that the introduced DNA was maintained as a plasmid in S. cellulosum So ce90. However, the plasmid pRP-GFP in the So ce90 transformants could not be detected using standard ethidium bromide staining, although a large amount of the plasmid pRP-GFP could be extracted from E. coli. This result suggested that the copy number became lower in the host strain. Low-copy-number plasmid vectors are sometimes useful for the cloning of a foreign gene, particularly when it codes for a toxic substance. So far, it has been difficult and complicated to introduce foreign DNA into S. cellulosum by conjugation. This bacterium is naturally multiresistant against most commonly used antibiotics and often, genetic methods established for one strain cannot be applied to others, even if they are phylogenetically closely related (12). Using biparental and triparental mating, Kopp et al. (11) has developed methodologies for DNA transfer from E. coli via conjugation to the strain So ce56, but they found that the same conjugation protocol was not applicable to So ce12. Thus, although pRPGFP had successfully transconjugated into So ce90 in our experiment, it was not unknown whether pRP-GFP could be used for to other S. cellulosum strains. Further research is needed to confirm this. Moreover, a worthwhile investigation concerned the transconjugant frequency of the system of gene transfer to S. cellulosum. In the first system, which was established by Jaoua et al. (9) with the vector pSUP2021 derived from pBR325, a plasmid with a Tn5 transposon, it was found that integrating DNA with regions of homology of less than 1000 bp could be extremely difficult because the recombination frequency of the host is very low for homology recombination. Obviously, without the occurrence of homology recombination, the transfer might be operable using the autonomously replicating plasmid pRP-GFP with smaller fragments. Furthermore, some attempts have been made to increase the transfer frequency using heat shock. Jaoua et al. (9) raised the frequency by a factor 10 by heating S. cellulosum cells at 50°C for 10 min. However, we found that the frequency of transposition did not change using the same manipulation technique, which is consistent with the result of Julien and Fehd (8). We found that mating time was closely related to the frequency. To obtain the highest frequency, it is necessary to avoid excessively long or short mating time. In this study, the GFP reporter system in S. cellulosum was also first established. GFP is the most recent and most popular reporter, since it has numerous advantages over other systems, being stable to temperature, pH, and denaturing agents. It is easily and nondestructively detected by illumination with blue or near-UV light and, unlike all other systems, requires no substrate. Furthermore, because the fluorescence of GFP is linearly proportional to the amount of the protein, it is an attractive reporter for monitoring in situ
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gene expression (21). S. cellulosum is difficult to engineer owing to the low efficiency of introducing DNA into it and the limited numbers of molecular tools and markers that have been developed for the organism (8, 9, 11). The stable expression of GFP in S. cellulosum was rapid and convenient for the conjugation screening of the transformants of the bacterium. Although the gene cluster responsible for the biosynthesis of epothilones in S. cellulosum So ce90 has been cloned and sequenced, the promoter of this operon is still unknown. Molnár et al. (13) considered that the promoter might lie in the stretch immediately preceding epoA in a 935-bp noncoding region. In our experiments, the comparison of the expression of gfp within pRP-GFP with that within pRK415GFP in S. cellulosum confirmed that the EpoPro fragment had the function of a promoter that could drive the expression of the gene in the bacterium. However, noweverHo GFP expression was detected in E. coli, which might be attributable to either the promoter not be recognized by E. coli RNA polymerase or other transcription factors required for the transcription. Further research on the properties of the EpoPro fragment as a promoter would be interesting. To date, no naturally occurring plasmids have been described in myxobacteria and no broad-host-range vectors are able to replicate in myxobacteria. Therefore, the work reported here will provide a powerful tool for isolating and analyzing the genes involved in epothilone metabolism. In this sense, this work may be a breakthrough in the genetics and molecular biology of S. cellulosum So ce90. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 30740474). We thank Shu-Sheng Yang of China Agricultural University for generously providing E. coli strain S17-1. We also thank Xu-Qing Chen of the Beijing Research Center of Agro-Biotechnology for helpful discussions and the critical reading of the manuscript.
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