JOURNAL
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Vol. 173, No. 20
0021-9193/91/206325-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Transformation System for Amycolatopsis (Nocardia) mediterranei: Direct Transformation of Mycelium with Plasmid DNA JERZY MADONt* AND RALF HUTTER Institute of Microbiology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland Received
5
April 1991/Accepted 12 July 1991
A new procedure for transformation of Amycolatopsis (Nocardia) mediterranei LBG A3136 was developed. The method makes use of polyethylene glycol and alkaline cations and enables direct transformation of the A. mediterranei mycelium with high efficiency: more than 106 transformants per ,ug of DNA were obtained. Transformation of A. mediterranei is stimulated by the ionophore antibiotic valinomycin and abolished by arsenate and p-chloromercuribenzenesulfonate. pMEA123, a vector based on the indigenous plasmid pMEA100 and containing the erythromycin resistance gene, was constructed.
Amycolatopsis (Nocardia) mediterranei LBG A3136 is a rifamycin-producing actinomycete (6). It contains the 23.7kb plasmid pMEA100 both in the integrated state and as an extrachromosomal element. pMEA100 elicits the lethal zygosis phenotype, is capable of site-specific excision and integration, and is subject to internal rearrangements (15, 20, 28). Further studies of the site-specific recombination and rearrangements of pMEA100 and the mechanism of biosynthesis of rifamycin would be greatly facilitated by the availability of a suitable method for transformation of A. mediterranei. Since none of the procedures used for transformation of streptomycetes and other actinomycetes was useful for transformation of A. mediterranei, we developed a new method enabling transformation of the strain with high efficiency. In addition, we constructed a vector based on the pMEA100 replicon and containing the erythromycin resistance gene.
MATERIALS AND METHODS
Bacterial strains and plasmids. The following A. mediterranei strains were used in this work: LBG A3136 (wild type [6]), LBG A3136/P (a plasmid-free derivative of LBG A3136 [20]), and LBG A3136/D (derivative of LBG A3136 containing a mixture of deletion forms of pMEA100). Plasmid pMEA52 was constructed by P. Zanella (27) on the basis of pIJlOl (8) and contains the thiostrepton, viomycin, and erythromycin resistance genes. Plasmid pMEA100 is an indigenous DNA element of A. mediterranei LBG A3136 (20). Constructs pMEA122 and pMEA123 are derivatives of pMEA100 and are described in detail in Results. Culture conditions. A. mediterranei strains were propagated in TS broth (2) and on yeast-malt extract-agar plates (22). Isolation of total cellular and plasmid DNAs. Total cellular and plasmid DNAs from A. mediterranei were isolated as described by Moretti et al. (20). Plasmid DNA was additionally purified by centrifugation in a CsCl gradient (17). The yields from isolations of pMEA122 and pMEA123 were very low, and the plasmid bands were hardly visible in the CsCl
gradients. The plasmids pMEA122 and pMEA123 were therefore precipitated directly from a fraction of the CsCl gradient as follows. To 1 ml of the CsCl gradient fraction containing the plasmid were added 3 ml of water, 0.3 ml of sodium acetate (pH 4.5), and 6.6 ml of absolute ethanol. The mixture was kept on ice for 30 min and then centrifuged in Eppendorf tubes at 13,000 rpm (Haereus table top centrifuge; Biofuge A) for 15 min at 4°C. The pellet was washed three times with 90% ethanol, dried in a vacuum, and then resuspended in buffer containing 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA. Electrophoresis and isolation of DNA fragments. Electrophoresis of DNA in agarose gels was carried out as described by Maniatis et al. (17). DNA fragments were isolated from agarose gels by adsorption onto an NA-45 DEAE-cellulose membrane (Schleicher and Schuell, Feldbach, Switzerland) as recommended by the supplier. Southern transfer and hybridization. Nick translation, Southern transfer, and hybridization were carried out as described by Maniatis et al. (17). Kodak XAR-5 X-ray films were used for autoradiography. Enzymes and labeled nucleotides. Restriction endonucleases were obtained from Boehringer (Mannheim, Germany), Pharmacia, or Promega Biotech. DNA polymerase I, DNase (RNase free), T4 DNA ligase, and calf intestine alkaline phosphatase were from Boehringer. Radioactive nucleoside triphosphates were obtained from Amersham. Cloning experiments. Cloning of DNA was carried out as described by Maniatis et al. (17). Transformation of the A. mediterranei mycelium. A. mediterranei LBG A3136/P was cultivated in TS broth for 42 to 50 h. One hundred milliliters of medium was added to a baffled 1-liter flask, which was subsequently inoculated with 0.1 ml of a suspension of a fully grown culture (see below) in glycerol and incubated in a orbital shaker at 150 rpm and 30°C. Aliquots (10 ml) of the culture were centrifuged in Sterilin tubes at 5,000 rpm for 15 min (Vismar table centrifuge; room temperature); two or three centrifugations with successively smaller volumes of medium were necessary to pellet the mycelium satisfactorily (4 to 6 g of wet mycelium were obtained from 100 ml of culture). Afterward the mycelium was resuspended in 4 ml of 20% glycerol and stored at -20°C. Directly before transformation, mycelial suspensions were thawed, washed three times in 2 volumes of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA), and resuspended in TE buffer (0.25 ml of TE per ml of glycerol
Corresponding author. t Present address: Institute of Organic Chemistry, University Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. *
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suspension for the standard procedure). Mycelium and transformation mixtures were prepared at room temperature. The standard transformation mixture (total volume, 0.2 ml) contained 0.05 ml of TE-mycelium suspension, 5 mM MgCl2, 0.625 M CsCl, 7.5 ,g of sonicated calf thymus DNA (Serva), 0.02 to 0.5 ,ug of plasmid DNA, and 35% polyethylene glycol (PEG 1000; Koch Light). To 50 [l of TEmycelium suspension were added 10 ,ul of 0.1 M MgCl2 31.3 ,u of 4 M CsCl, 1 ,u of calf thymus DNA, 1 to 5 RI of plasmid DNA, and TE buffer to final volume of 100 [lI. This was followed by the addition of 100 pAl of 70% (wt/vol) PEG in TE. After each constituent was added, the mixture was mixed thoroughly. The thorough mixing was especially important after the addition of PEG. The stock solution of calf thymus DNA was prepared in the following way. A 10-mg/ml solution (in TE buffer) of calf thymus DNA was sonicated until it had a very low viscosity, and it was then centrifuged in Eppendorf tubes at 13,000 rpm for 15 min (Haereus table top centrifuge; Biofuge A). The supernatant was incubated at 100°C for 20 min, cooled on ice, and stored at -200C. The transformation mixture was incubated at 30°C for 40 min and then at 42°C for 5 min and finally cooled to room temperature. Samples (2 to 50 ,u) of the transformation mixture were plated in 3 ml of overlay medium (containing R2L medium [24] and low-melting-point agarose; see below) on S27M agar plates containing 73.2 g of D-mannitol (Fluka), 5 g of peptone (Difco), 3 g of yeast extract (Difco), and 17 g of Bacto-Agar (Difco) per liter. In the case of selection by pock formation, 10 ,u of untreated A. mediterranei mycelium (glycerol suspension; see above) was added to the overlay medium along with the transformation mixture. If necessary, the transformation mixture was diluted with R2L medium or with P medium (2). The S27M agar plates were prepared on the day of transformation and dried in a laminarflow cabinet for 3 h. After the transformation mixture was plated, the plates were incubated for 5 to 10 days at 30°C. When transformants were selected by resistance against an antibiotic, 1 ml of the antibiotic (suspended in water) was overlaid on each plate after 16 to 20 h of incubation. The method of preparation of the R2L-agarose overlay medium was critical for the success of the transformation technique. To prepare 1 liter of the overlay medium, the following ingredients were added: 73.2 g of D-mannitol (Fluka), 0.25 g of K2S04, 10.1 g of MgCl2 6H20, 10 g of glucose, 0.1 g of Casamino Acids (Difco), 5 g of yeast extract (Difco), 100 ml of 0.25 M TES (pH 7.2), 80 ml of 3.68% (wt/vol) CaCl2 2H20, 10 ml of 0.5% (wt/vol) KH2PO4, 2 ml of trace elements solution (8), 20 ml of 10% (wt/vol) asparagine; 5 ml of 1 N NaOH, and 7 g of low-melting-point agarose (type VII; Sigma). Mannitol, K2S04, MgCl2, glucose, Casamino Acids, yeast extract, and low-melting-point agarose can be autoclaved together and stored at room temperature. The other ingredients must be autoclaved separately and added (in the following sequence: TES, CaCl2, KH2PO4, trace elements, asparagine, NaOH) to the main mixture shortly before use. NaOH must be added dropwise with vigorous mixing. NaOH added too fast caused the immediate appearance of a precipitate, which then dramatically decreased the transformation efficiency (data not shown). After the addition of NaOH, the medium has to be mixed intensively for about 30 s. Some precipitate may later appear in the medium, but it has no influence on the transformation efficiency. For plating, the transformation mixture should be added
J. BACTERIOL.
directly to the overlay medium (kept in a 42°C water bath), thus avoiding contact with any condensed water on the walls of the tube. This is especially important when small volumes of the transformation mixture are plated, because dilution of the transformed cells with water greatly decreases the number of transformants. RESULTS Direct transformation of the A. mediterranei mycelium: general remarks. In contrast to the laborious and sometimes critical preparation of protoplasts (an indispensable step in the established transformation procedures for actinomycetes), the mycelium of A. mediterranei used for transformation does not need any special preparation, only washing in TE buffer before use. Preincubation of mycelium with cations or with both cations and plasmid DNA decreased the number of transformants significantly (data not shown). Preincubation with cations and then with DNA turned out to be necessary for S. cerevisiae and Escherichia coli cells before PEG was added (9, 11). The highest number of A. mediterranei transformants was observed when the complete transformation mixture was incubated at 30°C for 40 to 60 min and then at 42°C for 5 min. Without these incubations (plating directly after addition of PEG), however, the transformation efficiency achieved was still high and amounted to about 50% of the maximum. The transformation mixture has to be plated in R2L-agarose overlay medium (see Materials and Methods). Direct plating of the transformation mixture or the use of other overlay media (8, 26) gave unsatisfactory results; no or very few transformants were obtained. The presence of mannitol in the basal S27M agar medium was absolutely necessary; without it no transformants were obtained. The times of drying and storage of the S27M agar plates (see Materials and Methods) also strongly affected the transformation efficiency. Drying for too short or too long a time and storing the S27M agar plates (at room temperature or at 4°C) for longer than 1 day reduced the number of transformants significantly (data not shown). The A. mediterranei transformants were selected by the lethal zygosis reaction (Ltz phenotype; pock formation [8]) or by resistance to antibiotics. To better visualize pocks, fresh A. mediterranei mycelium was added to the overlay medium (see Materials and Methods). Cells from more than 100 pocks were tested for their content of covalently closed circular DNA, and in all cases the plasmid used for transformation was found. Influence of cations and PEG. The presence of cations and PEG in the transformation mixture is absolutely necessary. Among the cations tested, potassium, cesium, and rubidium ions turned out to be most effective (Fig. 1B). K+ and Cs' ions showed the same optimal concentrations, i.e., 0.625 M, although the stimulation of transformation by Cs' ions was somewhat stronger. Surprisingly, the optimal concentration of the Rb+ cations was twice as high as that of K+ or Cs' cations (Fig 1B). In the presence of Na+ and Li' cations, only a few transformants were obtained. Magnesium ions strongly stimulated the transformation of A. mediterranei (Fig. 1A). This stimulation occurred, however, only in the presence of potassium, cesium, or rubidium ions (no transformants were observed without these alkaline cations). The addition of calcium ions prevented transformation in the presence of Mg2", K+, Cs', or Rb+ ions as well as without them. PEG is a necessary ingredient of the transformation mixture and exhibits its effect in a relatively narrow concentra-
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FIG. 1. Effect of salts, PEG, mycelium, and DNA concentrations on transformation of A. mediterranei. For details see Materials and Methods. (A) effect of magnesium ions; (B) effect of cesium (-) and rubidium (0) ions; (C) effect of PEG; (D) effect of the mycelium concentration (mycelium was obtained from A. mediterranei culture propagated for 50 h to the stationary phase; the optical density [OD] of this culture at 400 nm was 12.5; 1 unit of mycelium optical density therefore corresponds to the amount of mycelium obtained from 0.085 ml of culture); (E) effect of calf thymus DNA concentration; (F) effect of pMEA100 concentration.
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TABLE 1. Influence of valinomycin, arsenate, and p-CMBS on
transformation of A. mediterranei LBG A3136/Pa CsClIconcn (M) 0.250 0.250 0.250 0.250
0.625 0.625 0.625 0.625
Relative
Addition
transformation efficiency
Valinomycin (2 ,uM) Arsenate (50 mM) p-CMBS (0.1 mM)
41 373 0 0.2
None
None
Valinomycin (2 ,uM) Arsenate (50 mM) p-CMBS (0.1 mM)
100 239 0 0.5
a For clarity, the number of transformants obtained at the optimal concentration of CsCl (0.625 M) without the addition of any other factor has been taken as 100; the other data have been calculated in relation to this control value. Mycelium was preincubated at 30°C for 5 min with valinomycin, arsenate, or p-CMBS in the mixture containing cations and calf thymus DNA (see Materials and Methods). Then plasmid DNA and PEG 1000 were added, and the main incubation was started. With the added factors at the final concentrations shown in parentheses, no significant inhibition of the strain growth was observed.
tion range (Fig. 1C). Without PEG, no transformants were observed. Effect of mycelium and DNA concentration. The age of the mycelium and its concentration affected the transformation efficiency significantly. The best results were achieved when mycelium of A. mediterranei was harvested during the stationary phase. Transformation of the mycelium harvested in the exponential growth phase gave at least three times fewer transformants (data not shown). The addition of calf thymus DNA greatly stimulated transformation of A. mediterranei (Fig. 1E) and allowed the concentration of the plasmid DNA used for transformation to be kept at a low level (Fig. 1F). Without this carrier DNA, very few transformants were obtained. Effect of valinomycin, arsenate, and p-chloromercuribenzenesulfonate (p-CMBS). The ionophore antibiotic valinomycin exhibits strong affinity to alkaline cations K+, Cs', and Rb+ and greatly increases the permeability of membranes specifically to these ions (3, 14, 21). In contrast, valinomycin shows little affinity to Na+ and Li' cations (21). Since the K+, Cs', and Rb+ cations exhibited strong stimulation of transformation of A. mediterranei (in contrast to the Na+ and Li' cations), we decided to examine whether valinomycin could influence the efficiency of transformation of our strain. Valinomycin increases the transformation efficiency of A. mediterranei very effectively (Table 1). Its influence on transformation depends on the concentration of the salt. The greatest increase was observed at 0.25 M KCl and CsCl (an increase of more than ninefold). The number of transformants at 0.625 M KCl or CsCl (i.e., at the optimal concentration) was around twice as high with valinomycin than without it (Table 1). At the optimal concentration of RbCl (1.25 M), valinomycin also increased the transformation efficiency twofold. The effect of valinomycin at other concentrations of RbCl was not investigated. Valinomycin did not stimulate the transformation of A. mediterranei when Na+ or Li' cations were present in the transformation mixture instead of K+, Cs', or Rb+ cations (data not shown). Transformation of A. mediterranei is totally or nearly totally abolished by arsenate or p-CMBS, respectively (Table 1). Valinomycin could not counteract the effects of
arsenate and p-CMBS (data not shown). The reduction of transformation efficiency caused by arsenate and p-CMBS did not result from inhibition of cell growth, since the inhibitors were used in concentrations (Table 1) at which the cells still grew normally (after dilution by plating; data not shown). Sensitivity of A. mediterranei LBG A3136 to antibiotics. A. mediterranei LBG A3136 (wild type and the plasmid-free strain) was resistant to thiostrepton, viomycin, spectinomycin, kanamycin, neomycin, and adriamycin. However, no growth of the plasmid-free strain was observed in the presence of 10 ,ug of erythromycin, streptomycin, and chloramphenicol per ml, 25 p.g of hygromycin per ml, 100 ,ug of novobiocin per ml, or 200 ,ug of ampicillin per ml. With the exception of chloramphenicol, two- to fourfold higher concentrations of antibiotic were needed to inhibit the growth of the wild-type strain of A. mediterranei entirely. Vector construction for transformation of A. mediterranei. The indigenous plasmid of A. mediterranei, pMEA100, was used to construct a new vector. To reduce the size of pMEA100 (23.7 kb), we took advantage of the naturally occurring deletion forms of the plasmid in a subculture of A. mediterranei (13). pMEA100D (plasmid preparation containing pMEA100 together with its deletion forms; data not shown) was cut with Asp 718, and the plasmid fragments were ligated together with the 1.6-kb Asp 718 fragment from pMEA52 (27) containing the erythromycin resistance gene (4). The ligation mixture was then used to transform A. mediterranei LBG A3136/P (20). The transformation mixture was plated as described in Materials and Methods, and the transformants were selected on agar plates containing erythromycin (final concentration, 100 ,ug/ml). The erythromycin-resistant colonies were picked after 7 to 10 days of growth. After propagation on yeast-malt extract-agar plates and in TS broth containing erythromycin (100 jag/ml) (see Materials and Methods), the status of the plasmid DNA in each obtained clone was analyzed (isolation of total cellular DNA, digestion of DNA with Asp 718, electrophoresis in an agarose gel, Southern transfer, and hybridization with pMEA100 as a probe) (data not shown). The strain containing the smallest plasmid construct was used for the isolation of covalently closed circular DNA. The restriction map of the new plasmid, called pMEA122, is presented in Fig. 2B. In the next step, pMEA122 was cut with Sacl and the bigger fragment of 11.6 kb was isolated from an agarose gel. The ends of this fragment were ligated together, and the new construct was used for the transformation of A. mediterranei LBG A3136/P. Plating, selection, and isolation of covalently closed circular DNA were performed as described above. The detailed restriction map of the new plasmid, called pMEA123, is presented in Fig. 2C and D. The single SacI restriction site of pMEA123 can be used for cloning experiments. Further attempts at reducing the size of the last construct have not yet given satisfactory results. Transformation of A. mediterranei LBG A3136/P with different plasmids. Different streptomycete vectors were used for the transformation of A. mediterranei. The highest transformation efficiency was achieved with pMEA52 (Table 2). Plasmid pMZ11.1 (16) gave 0.5 x 104 transformants per ,ug of DNA, and after transformation with pJOE837 (1) only a few transformants were observed. No transformants were obtained when pIJ941, pIJ400, or pIJ405 (7, 8) was used. Growth of all the transformants on plates was extremely slow (3 to 4 weeks of incubation time was necessary before colonies could be picked), and no subsequent growth of the
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TRANSFORMATION SYSTEM FOR A. MEDITERRANEI
A
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B
Sad
I D
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i
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FIG. 2. Vector construction for transformation of A. mediterranei. Symbols: Eli, pMEA100 sequences; _, deleted erythromycin resistance gene (4); arrowhead in panel A, integration site (attP) (15). pMEA100;
sequences
of
El,
obtained clones was observed in liquid media. The transformants were not analyzed further. In contrast to the streptomycete vectors, the indigenous plasmid of A. mediterranei, pMEA100, gave high transformation efficiency (Table 2) and its transformants grew well TABLE 2. Transformation of A. mediterranei LBG A3136/P with different plasmidsa Vector
Size (kb)
Source of
Marker(s)
replication origin
Reference r source
Transformants per Lg of DNA
0.5 x 106b 27 7.3 Eryr, Thior, pIJ101 Vior 1.2 x 106 pMEA100 20 pMEA100 23.7 Ltz pMEA100 This work 0.4 x 106 pMEA123 11.6 Eryr
pMEA52
a For details see Materials and Methods. The pMEA52 and pMEA123 transformants were selected for resistance to erythromycin (Eryr; final concentration, 100 jig/ml) and the pMEA100 transformants were selected by the lethal zygosis reaction (Ltz, pock formation [8]). Thior, resistance to thiostrepton; Vior, resistance to viomycin. b Colonies of the pMEA52 transformants grew very slowly; at least 3 weeks of incubation was necessary before transformants could be picked for further experiments.
plates as well as in liquid media. High transformation efficiency was also observed after transformation of A. mediterranei with the new construct pMEA123, although the number of transformants obtained per microgram of plasmid DNA was three times smaller than that with pMEA100 (Table 2). pMEA100 and its natural deletion forms occurring in the pMEA100D preparation (see above) have the Ltz+ phenotype. pMEA100 (20) and pMEA100D therefore give rise to pocks when plasmid-carrying cells develop in a confluent lawn of cells lacking the plasmid. Pocks have been characterized as areas of reduced sporulation, increased (changed) pigmentation, and/or reduced growth resulting from the plasmid transfer (8). We observed significant differences between pocks obtained after transformation of the plasmidfree strain with pMEA100 and those obtained after transformation with pMEA100D (Fig. 3). Growth of cells into which plasmid molecules of the pMEA100D preparation had been transferred was strongly retarded (maybe some of the cells were even lysed), which caused the pMEA100D pocks to look like holes in the lawn. The visibility of the pMEA100D pocks increased during incubation of plates. In contrast, the pMEA100 pocks were much less distinct and became less on
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B
FIG. 3. Pocks produced on a lawn of the plasmid-free cells of A. mediterranei LBG A3136. (A) Section of a plate showing pocks obtained after transformation of the strain with pMEA100; (B) section of a plate showing pocks obtained after transformation of the plasmid-free strain with pMEA100D. Transformation and plating were carried out as described in Materials and Methods. The plates were incubated for 4 days at 30°C. Similar numbers of pocks were observed on both kinds of plates. For photography, the plates were illuminated from below.
visible and/or disappeared with time (they were overgrown by cells of the lawn). The difference between these two kinds of pocks was especially apparent when the plates were illuminated from below (Fig. 3). DISCUSSION In numerous techniques used for the transformation of streptomycetes and other actinomycetes, protoplasts are transformed with DNA in the presence of PEG. We tried unsuccessfully to transform protoplasts of A. mediterranei LBG A3136 obtained by the method of Schupp and Divers (24) and modifications of that method (data not shown) with different transformation techniques described earlier (8, 18, 26) and combinations thereof. Therefore we developed a procedure that allows direct transformation of mycelium of A. mediterranei LBG A3136 with plasmid DNA. This method is simple to use and guarantees high transformation efficiency. It can be used as an alternative method for transformation of other actinomycete strains when protoplast-PEG techniques do not give satisfactory results. We have not systematically examined the efficiency of the procedure described here for the transformation of other actinomycetes. It was found, however, that Nocardia sp. strain 239 could be transformed with high efficiency (25). No transformants have been obtained until now by transformation of Streptomyces lividans. The new transformation procedure includes treatment of mycelium with PEG as well as with potassium, cesium, or rubidium cations in high concentrations. The addition of magnesium ions and calf thymus DNA greatly increased the transformation efficiency. PEG has already been used successfully for direct transformation of prokaryotic and eukaryotic cells (9, 11). In the case of E. coli, the effect of PEG was reinforced by low concentrations of magnesium and potassium ions (2.5 and 12 mM, respectively [11]). The transformation of S. cerevisiae could be induced with sodium, potassium, cesium, rubidium, and lithium ions (9); with the exception of cesium cations, whose optimal con-
centration was 1 M, the ions were used in 0.1 to 0.2 M concentrations. Transformation of A. mediterranei is stimulated by the ionophore antibiotic valinomycin and abolished by arsenate, an inhibitor of oxidative phosphorylation (12), and by p-CMBS, which blocks functional -SH groups. The effect of valinomycin was observed only in the presence of potassium cesium, or rubidium ions. Valinomycin had no significant effect on the transformation of E. coli, whereas another ionophore, nigericin, strongly inhibited the transformation of this strain (23). Uptake of DNA by E. coli, which is induced by calcium ions, manifests great resistance to uncouplers of oxidative phosphorylation (23). Transformation of A. mediterranei with the streptomycete vectors did not give satisfactory results. Although high transformation efficiency was achieved (as in the case of pMEA52; see Results), the growth of transformants on plates was exceptionally slow and in liquid medium no growth was observed at all. The retarded growth on plates and lack of growth in liquid medium could result from incompatibility between the replicons of the streptomycete plasmids used and that of the A. mediterranei chromosome or may be caused by abortive transformation. However, such problems were not observed with another Amycolatopsis species, A. orientalis, which could be transformed with streptomycete vectors with high efficiency and whose transformants grew normally (18). On the other hand, very good results were obtained when pMEA100 was used for transformation of A. mediterranei. We therefore decided to construct a vector based on this indigenous plasmid. The constructed vector, called pMEA123, contains only 42% of the pMEA100 sequences, the erythromycin resistance gene, and a single SacI restriction site that can be used for cloning experiments. However, we consider the construction of pMEA123 only as the first step on the way to vector construction for transformation of A. mediterranei. Further attempts leading to the reduction of the plasmid size should be undertaken, and a second marker and additional single restriction sites should be introduced.
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An increase in the yield of pMEA123 is also desirable. The preliminary investigations in this direction showed that the low yield of isolation of this plasmid (only around 20 ,ug of pMEA123 was obtained from 1 liter of A. mediterranei culture) could be connected with the loss of some pMEA100 sequences during the vector construction (data not shown). Noticeable differences were found between pocks obtained after transformation of A. mediterranei with pMEA100 and pMEA100D. The pMEA100D pocks resembled phage plaques more than the actinomycete pocks described earlier (10, 20). When the cells from some pMEA100D pocks were picked and propagated further on agar plates, typical phage plaques were observed (data not shown). However, preliminary electron microscopic analysis of the contents on these plaques did not show intact phages, although we observed particles similar to phage heads and tails (data not shown). Typical plaques were also obtained when the A. mediterranei cells were transformed with the pMEA100 deletion forms isolated from the recipient strain after crossing of the wild-type A. mediterranei LBG A3136 (containing pMEA100) with the plasmid-free strain A. mediterranei T88 (19; data not shown). These data suggest that pMEA100 encodes some normally inactive phage functions that are unblocked during the transformation process and/or the conjugational transfer. On the basis of DNA sequence comparisons, Boccard et al. (5) concluded that the conjugative plasmid pSAM2 from Streptomyces ambofaciens is related to temperate bacteriophages. ACKNOWLEDGMENTS We thank Richard Plater for reading the manuscript. The project was supported by grants 08230 and 06278 from the Swiss Federal Institute of Technology and by grants 1231 and 1568 from the Swiss Committee for the Promotion of Research. REFERENCES 1. Altenbuchner, J. Unpublished data. 2. Baltz, R. H. 1978. Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration. J. Gen. Microbiol. 107:93-102. 3. Bhattacharyya, P., W. Epstein, and S. Silver. 1971. Valinomycin-induced uptake of potassium in membrane vesicles from E. coli. Proc. Nat. Acad. Sci. USA 68:1488-1492. 4. Bibb, M. J., G. R. Janssen, and J. M. Ward. 1986. Cloning and analysis of the promoter region of the erythromycin-resistance gene (erm E) of Streptomyces erythraeus. Gene 41:E357-E368. 5. Boccard, F., T. Smokvina, J.-L. Pernodet, A. Friedmann, and M. Guerineau. 1989. The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. EMBO J. 8:973-980. 6. Ghisalba, 0., J. A. L. Auden, T. Schupp, and J. Nuesch. 1984. The rifamycins: properties, biosynthesis, and fermentation, p. 281-327. In E. J. Vandamme (ed.), Biotechnology of industrial antibiotics. Marcel Dekker, New York. 7. Hintermann, G., R. Crameri, M. Vogtli, and R. Hutter. 1984. Steptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase. Mol. Gen. Genet. 196:513-520.
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8. Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J. Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. 1985. Genetic manipulation of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich, England. 9. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 152:163-168. 10. Kieser, T., D. A. Hopwood, H. M. Wright, and C. J. Thompson. 1982. pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol. Gen. Genet. 185:223-238. 11. Klebe, R. J., J. V. Harris, Z. D. Sharp, and M. G. Douglas. 1983. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333-341. 12. Klein, W. L., and P. D. Boyer. 1972. Energization of active transport by E. coli. J. Biol. Chem. 247:7257-7265. 13. La Roche, S. Unpublished data. 14. Lombardi, F. J., J. P. Reeves, and H. R. Kaback. 1973. Mechanism of active transport in isolated bacterial membrane vesicles. XIII. Valinomycin-induced rubidium transport. J. Biol. Chem. 248:3551-3565. 15. Madon, J., P. Moretti, and R. Hutter. 1987. Site-specific integration and excision of pMEA100 in Nocardia mediterranei. Mol. Gen. Genet. 209:257-264. 16. Malpartida, F., M. Zalacain, A. Jimenez, and J. Davies. 1983. Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. Biochem. Biophys. Res. Commun. 117:6-12. 17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Matsushima, P., M. A. McHenny, and R. H. Baltz. 1987. Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids. J. Bacteriol. 169:2298-3200. 19. Moretti, P. Personal communication. 20. Moretti, P., G. Hintermann, and R. Hutter. 1985. Isolation and characterization of an extrachromosomal element from Nocardia mediterranei. Plasmid 14:126-133. 21. Ovchinnikov, Y. A. 1979. Physico-chemical basis of ion transport through biological membranes: ionophores and ion channels. Eur. J. Biochem. 94:321-336. 22. Pridham, T. G., P. Anderson, C. Foley, L. A. Lindenfelser, C. W. Hesseltine, and R. G. Benedict. 1956/1957. A selection of media for maintenance and taxonomic study of Streptomyces. Antibiot. Annu., p. 947-952. 23. Sabelnikov, A. G., and I. V. Domaradsky. 1981. Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2'-treated Escherichia coli cells. J. Bacteriol. 146:
435-443. 24. Schupp, T., and M. Divers. 1986. Protoplast preparation and regeneration in Nocardia mediterranei. FEMS Microbiol. Lett.
36:159-162. 25. VrUbloed, J. W. Personal communication. 26. Yamamoto, H., and K. H. Maurer. 1986. Transformation of Streptomyces erythraeus. J. Antibiot. 39:1304-1313. 27. Zanella, P. Unpublished data. 28. Zhu, B., J. Madon, A. Hiusler, and R. Hutter. 1990. Amplification on the Amycolatopsis (Nocardia) mediterranei plasmid pMEA100: sequence similarities to actinomycetes att sites. Plasmid 24:132-142.
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0021-9193/91/206325-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Transformation System for Amycolatopsis (Nocardia) mediterranei: Direct Transformation of Mycelium with Plasmid DNA JERZY MADONt* AND RALF HUTTER Institute of Microbiology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland Received
5
April 1991/Accepted 12 July 1991
A new procedure for transformation of Amycolatopsis (Nocardia) mediterranei LBG A3136 was developed. The method makes use of polyethylene glycol and alkaline cations and enables direct transformation of the A. mediterranei mycelium with high efficiency: more than 106 transformants per ,ug of DNA were obtained. Transformation of A. mediterranei is stimulated by the ionophore antibiotic valinomycin and abolished by arsenate and p-chloromercuribenzenesulfonate. pMEA123, a vector based on the indigenous plasmid pMEA100 and containing the erythromycin resistance gene, was constructed.
Amycolatopsis (Nocardia) mediterranei LBG A3136 is a rifamycin-producing actinomycete (6). It contains the 23.7kb plasmid pMEA100 both in the integrated state and as an extrachromosomal element. pMEA100 elicits the lethal zygosis phenotype, is capable of site-specific excision and integration, and is subject to internal rearrangements (15, 20, 28). Further studies of the site-specific recombination and rearrangements of pMEA100 and the mechanism of biosynthesis of rifamycin would be greatly facilitated by the availability of a suitable method for transformation of A. mediterranei. Since none of the procedures used for transformation of streptomycetes and other actinomycetes was useful for transformation of A. mediterranei, we developed a new method enabling transformation of the strain with high efficiency. In addition, we constructed a vector based on the pMEA100 replicon and containing the erythromycin resistance gene.
MATERIALS AND METHODS
Bacterial strains and plasmids. The following A. mediterranei strains were used in this work: LBG A3136 (wild type [6]), LBG A3136/P (a plasmid-free derivative of LBG A3136 [20]), and LBG A3136/D (derivative of LBG A3136 containing a mixture of deletion forms of pMEA100). Plasmid pMEA52 was constructed by P. Zanella (27) on the basis of pIJlOl (8) and contains the thiostrepton, viomycin, and erythromycin resistance genes. Plasmid pMEA100 is an indigenous DNA element of A. mediterranei LBG A3136 (20). Constructs pMEA122 and pMEA123 are derivatives of pMEA100 and are described in detail in Results. Culture conditions. A. mediterranei strains were propagated in TS broth (2) and on yeast-malt extract-agar plates (22). Isolation of total cellular and plasmid DNAs. Total cellular and plasmid DNAs from A. mediterranei were isolated as described by Moretti et al. (20). Plasmid DNA was additionally purified by centrifugation in a CsCl gradient (17). The yields from isolations of pMEA122 and pMEA123 were very low, and the plasmid bands were hardly visible in the CsCl
gradients. The plasmids pMEA122 and pMEA123 were therefore precipitated directly from a fraction of the CsCl gradient as follows. To 1 ml of the CsCl gradient fraction containing the plasmid were added 3 ml of water, 0.3 ml of sodium acetate (pH 4.5), and 6.6 ml of absolute ethanol. The mixture was kept on ice for 30 min and then centrifuged in Eppendorf tubes at 13,000 rpm (Haereus table top centrifuge; Biofuge A) for 15 min at 4°C. The pellet was washed three times with 90% ethanol, dried in a vacuum, and then resuspended in buffer containing 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA. Electrophoresis and isolation of DNA fragments. Electrophoresis of DNA in agarose gels was carried out as described by Maniatis et al. (17). DNA fragments were isolated from agarose gels by adsorption onto an NA-45 DEAE-cellulose membrane (Schleicher and Schuell, Feldbach, Switzerland) as recommended by the supplier. Southern transfer and hybridization. Nick translation, Southern transfer, and hybridization were carried out as described by Maniatis et al. (17). Kodak XAR-5 X-ray films were used for autoradiography. Enzymes and labeled nucleotides. Restriction endonucleases were obtained from Boehringer (Mannheim, Germany), Pharmacia, or Promega Biotech. DNA polymerase I, DNase (RNase free), T4 DNA ligase, and calf intestine alkaline phosphatase were from Boehringer. Radioactive nucleoside triphosphates were obtained from Amersham. Cloning experiments. Cloning of DNA was carried out as described by Maniatis et al. (17). Transformation of the A. mediterranei mycelium. A. mediterranei LBG A3136/P was cultivated in TS broth for 42 to 50 h. One hundred milliliters of medium was added to a baffled 1-liter flask, which was subsequently inoculated with 0.1 ml of a suspension of a fully grown culture (see below) in glycerol and incubated in a orbital shaker at 150 rpm and 30°C. Aliquots (10 ml) of the culture were centrifuged in Sterilin tubes at 5,000 rpm for 15 min (Vismar table centrifuge; room temperature); two or three centrifugations with successively smaller volumes of medium were necessary to pellet the mycelium satisfactorily (4 to 6 g of wet mycelium were obtained from 100 ml of culture). Afterward the mycelium was resuspended in 4 ml of 20% glycerol and stored at -20°C. Directly before transformation, mycelial suspensions were thawed, washed three times in 2 volumes of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA), and resuspended in TE buffer (0.25 ml of TE per ml of glycerol
Corresponding author. t Present address: Institute of Organic Chemistry, University Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. *
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suspension for the standard procedure). Mycelium and transformation mixtures were prepared at room temperature. The standard transformation mixture (total volume, 0.2 ml) contained 0.05 ml of TE-mycelium suspension, 5 mM MgCl2, 0.625 M CsCl, 7.5 ,g of sonicated calf thymus DNA (Serva), 0.02 to 0.5 ,ug of plasmid DNA, and 35% polyethylene glycol (PEG 1000; Koch Light). To 50 [l of TEmycelium suspension were added 10 ,ul of 0.1 M MgCl2 31.3 ,u of 4 M CsCl, 1 ,u of calf thymus DNA, 1 to 5 RI of plasmid DNA, and TE buffer to final volume of 100 [lI. This was followed by the addition of 100 pAl of 70% (wt/vol) PEG in TE. After each constituent was added, the mixture was mixed thoroughly. The thorough mixing was especially important after the addition of PEG. The stock solution of calf thymus DNA was prepared in the following way. A 10-mg/ml solution (in TE buffer) of calf thymus DNA was sonicated until it had a very low viscosity, and it was then centrifuged in Eppendorf tubes at 13,000 rpm for 15 min (Haereus table top centrifuge; Biofuge A). The supernatant was incubated at 100°C for 20 min, cooled on ice, and stored at -200C. The transformation mixture was incubated at 30°C for 40 min and then at 42°C for 5 min and finally cooled to room temperature. Samples (2 to 50 ,u) of the transformation mixture were plated in 3 ml of overlay medium (containing R2L medium [24] and low-melting-point agarose; see below) on S27M agar plates containing 73.2 g of D-mannitol (Fluka), 5 g of peptone (Difco), 3 g of yeast extract (Difco), and 17 g of Bacto-Agar (Difco) per liter. In the case of selection by pock formation, 10 ,u of untreated A. mediterranei mycelium (glycerol suspension; see above) was added to the overlay medium along with the transformation mixture. If necessary, the transformation mixture was diluted with R2L medium or with P medium (2). The S27M agar plates were prepared on the day of transformation and dried in a laminarflow cabinet for 3 h. After the transformation mixture was plated, the plates were incubated for 5 to 10 days at 30°C. When transformants were selected by resistance against an antibiotic, 1 ml of the antibiotic (suspended in water) was overlaid on each plate after 16 to 20 h of incubation. The method of preparation of the R2L-agarose overlay medium was critical for the success of the transformation technique. To prepare 1 liter of the overlay medium, the following ingredients were added: 73.2 g of D-mannitol (Fluka), 0.25 g of K2S04, 10.1 g of MgCl2 6H20, 10 g of glucose, 0.1 g of Casamino Acids (Difco), 5 g of yeast extract (Difco), 100 ml of 0.25 M TES (pH 7.2), 80 ml of 3.68% (wt/vol) CaCl2 2H20, 10 ml of 0.5% (wt/vol) KH2PO4, 2 ml of trace elements solution (8), 20 ml of 10% (wt/vol) asparagine; 5 ml of 1 N NaOH, and 7 g of low-melting-point agarose (type VII; Sigma). Mannitol, K2S04, MgCl2, glucose, Casamino Acids, yeast extract, and low-melting-point agarose can be autoclaved together and stored at room temperature. The other ingredients must be autoclaved separately and added (in the following sequence: TES, CaCl2, KH2PO4, trace elements, asparagine, NaOH) to the main mixture shortly before use. NaOH must be added dropwise with vigorous mixing. NaOH added too fast caused the immediate appearance of a precipitate, which then dramatically decreased the transformation efficiency (data not shown). After the addition of NaOH, the medium has to be mixed intensively for about 30 s. Some precipitate may later appear in the medium, but it has no influence on the transformation efficiency. For plating, the transformation mixture should be added
J. BACTERIOL.
directly to the overlay medium (kept in a 42°C water bath), thus avoiding contact with any condensed water on the walls of the tube. This is especially important when small volumes of the transformation mixture are plated, because dilution of the transformed cells with water greatly decreases the number of transformants. RESULTS Direct transformation of the A. mediterranei mycelium: general remarks. In contrast to the laborious and sometimes critical preparation of protoplasts (an indispensable step in the established transformation procedures for actinomycetes), the mycelium of A. mediterranei used for transformation does not need any special preparation, only washing in TE buffer before use. Preincubation of mycelium with cations or with both cations and plasmid DNA decreased the number of transformants significantly (data not shown). Preincubation with cations and then with DNA turned out to be necessary for S. cerevisiae and Escherichia coli cells before PEG was added (9, 11). The highest number of A. mediterranei transformants was observed when the complete transformation mixture was incubated at 30°C for 40 to 60 min and then at 42°C for 5 min. Without these incubations (plating directly after addition of PEG), however, the transformation efficiency achieved was still high and amounted to about 50% of the maximum. The transformation mixture has to be plated in R2L-agarose overlay medium (see Materials and Methods). Direct plating of the transformation mixture or the use of other overlay media (8, 26) gave unsatisfactory results; no or very few transformants were obtained. The presence of mannitol in the basal S27M agar medium was absolutely necessary; without it no transformants were obtained. The times of drying and storage of the S27M agar plates (see Materials and Methods) also strongly affected the transformation efficiency. Drying for too short or too long a time and storing the S27M agar plates (at room temperature or at 4°C) for longer than 1 day reduced the number of transformants significantly (data not shown). The A. mediterranei transformants were selected by the lethal zygosis reaction (Ltz phenotype; pock formation [8]) or by resistance to antibiotics. To better visualize pocks, fresh A. mediterranei mycelium was added to the overlay medium (see Materials and Methods). Cells from more than 100 pocks were tested for their content of covalently closed circular DNA, and in all cases the plasmid used for transformation was found. Influence of cations and PEG. The presence of cations and PEG in the transformation mixture is absolutely necessary. Among the cations tested, potassium, cesium, and rubidium ions turned out to be most effective (Fig. 1B). K+ and Cs' ions showed the same optimal concentrations, i.e., 0.625 M, although the stimulation of transformation by Cs' ions was somewhat stronger. Surprisingly, the optimal concentration of the Rb+ cations was twice as high as that of K+ or Cs' cations (Fig 1B). In the presence of Na+ and Li' cations, only a few transformants were obtained. Magnesium ions strongly stimulated the transformation of A. mediterranei (Fig. 1A). This stimulation occurred, however, only in the presence of potassium, cesium, or rubidium ions (no transformants were observed without these alkaline cations). The addition of calcium ions prevented transformation in the presence of Mg2", K+, Cs', or Rb+ ions as well as without them. PEG is a necessary ingredient of the transformation mixture and exhibits its effect in a relatively narrow concentra-
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FIG. 1. Effect of salts, PEG, mycelium, and DNA concentrations on transformation of A. mediterranei. For details see Materials and Methods. (A) effect of magnesium ions; (B) effect of cesium (-) and rubidium (0) ions; (C) effect of PEG; (D) effect of the mycelium concentration (mycelium was obtained from A. mediterranei culture propagated for 50 h to the stationary phase; the optical density [OD] of this culture at 400 nm was 12.5; 1 unit of mycelium optical density therefore corresponds to the amount of mycelium obtained from 0.085 ml of culture); (E) effect of calf thymus DNA concentration; (F) effect of pMEA100 concentration.
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TABLE 1. Influence of valinomycin, arsenate, and p-CMBS on
transformation of A. mediterranei LBG A3136/Pa CsClIconcn (M) 0.250 0.250 0.250 0.250
0.625 0.625 0.625 0.625
Relative
Addition
transformation efficiency
Valinomycin (2 ,uM) Arsenate (50 mM) p-CMBS (0.1 mM)
41 373 0 0.2
None
None
Valinomycin (2 ,uM) Arsenate (50 mM) p-CMBS (0.1 mM)
100 239 0 0.5
a For clarity, the number of transformants obtained at the optimal concentration of CsCl (0.625 M) without the addition of any other factor has been taken as 100; the other data have been calculated in relation to this control value. Mycelium was preincubated at 30°C for 5 min with valinomycin, arsenate, or p-CMBS in the mixture containing cations and calf thymus DNA (see Materials and Methods). Then plasmid DNA and PEG 1000 were added, and the main incubation was started. With the added factors at the final concentrations shown in parentheses, no significant inhibition of the strain growth was observed.
tion range (Fig. 1C). Without PEG, no transformants were observed. Effect of mycelium and DNA concentration. The age of the mycelium and its concentration affected the transformation efficiency significantly. The best results were achieved when mycelium of A. mediterranei was harvested during the stationary phase. Transformation of the mycelium harvested in the exponential growth phase gave at least three times fewer transformants (data not shown). The addition of calf thymus DNA greatly stimulated transformation of A. mediterranei (Fig. 1E) and allowed the concentration of the plasmid DNA used for transformation to be kept at a low level (Fig. 1F). Without this carrier DNA, very few transformants were obtained. Effect of valinomycin, arsenate, and p-chloromercuribenzenesulfonate (p-CMBS). The ionophore antibiotic valinomycin exhibits strong affinity to alkaline cations K+, Cs', and Rb+ and greatly increases the permeability of membranes specifically to these ions (3, 14, 21). In contrast, valinomycin shows little affinity to Na+ and Li' cations (21). Since the K+, Cs', and Rb+ cations exhibited strong stimulation of transformation of A. mediterranei (in contrast to the Na+ and Li' cations), we decided to examine whether valinomycin could influence the efficiency of transformation of our strain. Valinomycin increases the transformation efficiency of A. mediterranei very effectively (Table 1). Its influence on transformation depends on the concentration of the salt. The greatest increase was observed at 0.25 M KCl and CsCl (an increase of more than ninefold). The number of transformants at 0.625 M KCl or CsCl (i.e., at the optimal concentration) was around twice as high with valinomycin than without it (Table 1). At the optimal concentration of RbCl (1.25 M), valinomycin also increased the transformation efficiency twofold. The effect of valinomycin at other concentrations of RbCl was not investigated. Valinomycin did not stimulate the transformation of A. mediterranei when Na+ or Li' cations were present in the transformation mixture instead of K+, Cs', or Rb+ cations (data not shown). Transformation of A. mediterranei is totally or nearly totally abolished by arsenate or p-CMBS, respectively (Table 1). Valinomycin could not counteract the effects of
arsenate and p-CMBS (data not shown). The reduction of transformation efficiency caused by arsenate and p-CMBS did not result from inhibition of cell growth, since the inhibitors were used in concentrations (Table 1) at which the cells still grew normally (after dilution by plating; data not shown). Sensitivity of A. mediterranei LBG A3136 to antibiotics. A. mediterranei LBG A3136 (wild type and the plasmid-free strain) was resistant to thiostrepton, viomycin, spectinomycin, kanamycin, neomycin, and adriamycin. However, no growth of the plasmid-free strain was observed in the presence of 10 ,ug of erythromycin, streptomycin, and chloramphenicol per ml, 25 p.g of hygromycin per ml, 100 ,ug of novobiocin per ml, or 200 ,ug of ampicillin per ml. With the exception of chloramphenicol, two- to fourfold higher concentrations of antibiotic were needed to inhibit the growth of the wild-type strain of A. mediterranei entirely. Vector construction for transformation of A. mediterranei. The indigenous plasmid of A. mediterranei, pMEA100, was used to construct a new vector. To reduce the size of pMEA100 (23.7 kb), we took advantage of the naturally occurring deletion forms of the plasmid in a subculture of A. mediterranei (13). pMEA100D (plasmid preparation containing pMEA100 together with its deletion forms; data not shown) was cut with Asp 718, and the plasmid fragments were ligated together with the 1.6-kb Asp 718 fragment from pMEA52 (27) containing the erythromycin resistance gene (4). The ligation mixture was then used to transform A. mediterranei LBG A3136/P (20). The transformation mixture was plated as described in Materials and Methods, and the transformants were selected on agar plates containing erythromycin (final concentration, 100 ,ug/ml). The erythromycin-resistant colonies were picked after 7 to 10 days of growth. After propagation on yeast-malt extract-agar plates and in TS broth containing erythromycin (100 jag/ml) (see Materials and Methods), the status of the plasmid DNA in each obtained clone was analyzed (isolation of total cellular DNA, digestion of DNA with Asp 718, electrophoresis in an agarose gel, Southern transfer, and hybridization with pMEA100 as a probe) (data not shown). The strain containing the smallest plasmid construct was used for the isolation of covalently closed circular DNA. The restriction map of the new plasmid, called pMEA122, is presented in Fig. 2B. In the next step, pMEA122 was cut with Sacl and the bigger fragment of 11.6 kb was isolated from an agarose gel. The ends of this fragment were ligated together, and the new construct was used for the transformation of A. mediterranei LBG A3136/P. Plating, selection, and isolation of covalently closed circular DNA were performed as described above. The detailed restriction map of the new plasmid, called pMEA123, is presented in Fig. 2C and D. The single SacI restriction site of pMEA123 can be used for cloning experiments. Further attempts at reducing the size of the last construct have not yet given satisfactory results. Transformation of A. mediterranei LBG A3136/P with different plasmids. Different streptomycete vectors were used for the transformation of A. mediterranei. The highest transformation efficiency was achieved with pMEA52 (Table 2). Plasmid pMZ11.1 (16) gave 0.5 x 104 transformants per ,ug of DNA, and after transformation with pJOE837 (1) only a few transformants were observed. No transformants were obtained when pIJ941, pIJ400, or pIJ405 (7, 8) was used. Growth of all the transformants on plates was extremely slow (3 to 4 weeks of incubation time was necessary before colonies could be picked), and no subsequent growth of the
VOL. 173, 1991
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A
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B
Sad
I D
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i
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FIG. 2. Vector construction for transformation of A. mediterranei. Symbols: Eli, pMEA100 sequences; _, deleted erythromycin resistance gene (4); arrowhead in panel A, integration site (attP) (15). pMEA100;
sequences
of
El,
obtained clones was observed in liquid media. The transformants were not analyzed further. In contrast to the streptomycete vectors, the indigenous plasmid of A. mediterranei, pMEA100, gave high transformation efficiency (Table 2) and its transformants grew well TABLE 2. Transformation of A. mediterranei LBG A3136/P with different plasmidsa Vector
Size (kb)
Source of
Marker(s)
replication origin
Reference r source
Transformants per Lg of DNA
0.5 x 106b 27 7.3 Eryr, Thior, pIJ101 Vior 1.2 x 106 pMEA100 20 pMEA100 23.7 Ltz pMEA100 This work 0.4 x 106 pMEA123 11.6 Eryr
pMEA52
a For details see Materials and Methods. The pMEA52 and pMEA123 transformants were selected for resistance to erythromycin (Eryr; final concentration, 100 jig/ml) and the pMEA100 transformants were selected by the lethal zygosis reaction (Ltz, pock formation [8]). Thior, resistance to thiostrepton; Vior, resistance to viomycin. b Colonies of the pMEA52 transformants grew very slowly; at least 3 weeks of incubation was necessary before transformants could be picked for further experiments.
plates as well as in liquid media. High transformation efficiency was also observed after transformation of A. mediterranei with the new construct pMEA123, although the number of transformants obtained per microgram of plasmid DNA was three times smaller than that with pMEA100 (Table 2). pMEA100 and its natural deletion forms occurring in the pMEA100D preparation (see above) have the Ltz+ phenotype. pMEA100 (20) and pMEA100D therefore give rise to pocks when plasmid-carrying cells develop in a confluent lawn of cells lacking the plasmid. Pocks have been characterized as areas of reduced sporulation, increased (changed) pigmentation, and/or reduced growth resulting from the plasmid transfer (8). We observed significant differences between pocks obtained after transformation of the plasmidfree strain with pMEA100 and those obtained after transformation with pMEA100D (Fig. 3). Growth of cells into which plasmid molecules of the pMEA100D preparation had been transferred was strongly retarded (maybe some of the cells were even lysed), which caused the pMEA100D pocks to look like holes in the lawn. The visibility of the pMEA100D pocks increased during incubation of plates. In contrast, the pMEA100 pocks were much less distinct and became less on
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B
FIG. 3. Pocks produced on a lawn of the plasmid-free cells of A. mediterranei LBG A3136. (A) Section of a plate showing pocks obtained after transformation of the strain with pMEA100; (B) section of a plate showing pocks obtained after transformation of the plasmid-free strain with pMEA100D. Transformation and plating were carried out as described in Materials and Methods. The plates were incubated for 4 days at 30°C. Similar numbers of pocks were observed on both kinds of plates. For photography, the plates were illuminated from below.
visible and/or disappeared with time (they were overgrown by cells of the lawn). The difference between these two kinds of pocks was especially apparent when the plates were illuminated from below (Fig. 3). DISCUSSION In numerous techniques used for the transformation of streptomycetes and other actinomycetes, protoplasts are transformed with DNA in the presence of PEG. We tried unsuccessfully to transform protoplasts of A. mediterranei LBG A3136 obtained by the method of Schupp and Divers (24) and modifications of that method (data not shown) with different transformation techniques described earlier (8, 18, 26) and combinations thereof. Therefore we developed a procedure that allows direct transformation of mycelium of A. mediterranei LBG A3136 with plasmid DNA. This method is simple to use and guarantees high transformation efficiency. It can be used as an alternative method for transformation of other actinomycete strains when protoplast-PEG techniques do not give satisfactory results. We have not systematically examined the efficiency of the procedure described here for the transformation of other actinomycetes. It was found, however, that Nocardia sp. strain 239 could be transformed with high efficiency (25). No transformants have been obtained until now by transformation of Streptomyces lividans. The new transformation procedure includes treatment of mycelium with PEG as well as with potassium, cesium, or rubidium cations in high concentrations. The addition of magnesium ions and calf thymus DNA greatly increased the transformation efficiency. PEG has already been used successfully for direct transformation of prokaryotic and eukaryotic cells (9, 11). In the case of E. coli, the effect of PEG was reinforced by low concentrations of magnesium and potassium ions (2.5 and 12 mM, respectively [11]). The transformation of S. cerevisiae could be induced with sodium, potassium, cesium, rubidium, and lithium ions (9); with the exception of cesium cations, whose optimal con-
centration was 1 M, the ions were used in 0.1 to 0.2 M concentrations. Transformation of A. mediterranei is stimulated by the ionophore antibiotic valinomycin and abolished by arsenate, an inhibitor of oxidative phosphorylation (12), and by p-CMBS, which blocks functional -SH groups. The effect of valinomycin was observed only in the presence of potassium cesium, or rubidium ions. Valinomycin had no significant effect on the transformation of E. coli, whereas another ionophore, nigericin, strongly inhibited the transformation of this strain (23). Uptake of DNA by E. coli, which is induced by calcium ions, manifests great resistance to uncouplers of oxidative phosphorylation (23). Transformation of A. mediterranei with the streptomycete vectors did not give satisfactory results. Although high transformation efficiency was achieved (as in the case of pMEA52; see Results), the growth of transformants on plates was exceptionally slow and in liquid medium no growth was observed at all. The retarded growth on plates and lack of growth in liquid medium could result from incompatibility between the replicons of the streptomycete plasmids used and that of the A. mediterranei chromosome or may be caused by abortive transformation. However, such problems were not observed with another Amycolatopsis species, A. orientalis, which could be transformed with streptomycete vectors with high efficiency and whose transformants grew normally (18). On the other hand, very good results were obtained when pMEA100 was used for transformation of A. mediterranei. We therefore decided to construct a vector based on this indigenous plasmid. The constructed vector, called pMEA123, contains only 42% of the pMEA100 sequences, the erythromycin resistance gene, and a single SacI restriction site that can be used for cloning experiments. However, we consider the construction of pMEA123 only as the first step on the way to vector construction for transformation of A. mediterranei. Further attempts leading to the reduction of the plasmid size should be undertaken, and a second marker and additional single restriction sites should be introduced.
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TRANSFORMATION SYSTEM FOR A. MEDITERRANEI
An increase in the yield of pMEA123 is also desirable. The preliminary investigations in this direction showed that the low yield of isolation of this plasmid (only around 20 ,ug of pMEA123 was obtained from 1 liter of A. mediterranei culture) could be connected with the loss of some pMEA100 sequences during the vector construction (data not shown). Noticeable differences were found between pocks obtained after transformation of A. mediterranei with pMEA100 and pMEA100D. The pMEA100D pocks resembled phage plaques more than the actinomycete pocks described earlier (10, 20). When the cells from some pMEA100D pocks were picked and propagated further on agar plates, typical phage plaques were observed (data not shown). However, preliminary electron microscopic analysis of the contents on these plaques did not show intact phages, although we observed particles similar to phage heads and tails (data not shown). Typical plaques were also obtained when the A. mediterranei cells were transformed with the pMEA100 deletion forms isolated from the recipient strain after crossing of the wild-type A. mediterranei LBG A3136 (containing pMEA100) with the plasmid-free strain A. mediterranei T88 (19; data not shown). These data suggest that pMEA100 encodes some normally inactive phage functions that are unblocked during the transformation process and/or the conjugational transfer. On the basis of DNA sequence comparisons, Boccard et al. (5) concluded that the conjugative plasmid pSAM2 from Streptomyces ambofaciens is related to temperate bacteriophages. ACKNOWLEDGMENTS We thank Richard Plater for reading the manuscript. The project was supported by grants 08230 and 06278 from the Swiss Federal Institute of Technology and by grants 1231 and 1568 from the Swiss Committee for the Promotion of Research. REFERENCES 1. Altenbuchner, J. Unpublished data. 2. Baltz, R. H. 1978. Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration. J. Gen. Microbiol. 107:93-102. 3. Bhattacharyya, P., W. Epstein, and S. Silver. 1971. Valinomycin-induced uptake of potassium in membrane vesicles from E. coli. Proc. Nat. Acad. Sci. USA 68:1488-1492. 4. Bibb, M. J., G. R. Janssen, and J. M. Ward. 1986. Cloning and analysis of the promoter region of the erythromycin-resistance gene (erm E) of Streptomyces erythraeus. Gene 41:E357-E368. 5. Boccard, F., T. Smokvina, J.-L. Pernodet, A. Friedmann, and M. Guerineau. 1989. The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. EMBO J. 8:973-980. 6. Ghisalba, 0., J. A. L. Auden, T. Schupp, and J. Nuesch. 1984. The rifamycins: properties, biosynthesis, and fermentation, p. 281-327. In E. J. Vandamme (ed.), Biotechnology of industrial antibiotics. Marcel Dekker, New York. 7. Hintermann, G., R. Crameri, M. Vogtli, and R. Hutter. 1984. Steptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase. Mol. Gen. Genet. 196:513-520.
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