Journal of Microbiological Methods 53 (2003) 401 – 403 www.elsevier.com/locate/jmicmeth
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Improved PCR-based method for the direct screening of Streptomyces transformants W. Van Dessel, L. Van Mellaert, N. Geukens, J. Anne´ * Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Louvain, Belgium Received 10 July 2002; received in revised form 16 July 2002; accepted 4 November 2002
Abstract Streptomycetes are attractive microorganisms because of their high secretion capacity and for the production of secondary metabolites. We report the improvement of a PCR-based method for screening of solid media-grown Streptomyces transformants. By resuspending mycelium into 2.5 – 10% dimethyl sulfoxide (DMSO), extensive manipulation prior to PCR could be avoided, thus significantly reducing screening time. Results proved to be both reliable and specific. D 2002 Elsevier Science B.V. All rights reserved. Keywords: PCR screening; Transformants; Streptomyces
Since several decades, Streptomyces spp. are widely being used as a source for therapeutically important natural compounds. For example, more than 60% of the natural antibiotics are produced by these soil microorganisms. Nowadays, these bacteria are also becoming attractive tools for the production of heterologous proteins of biopharmaceutical and industrial importance (Gilbert et al., 1995; Hopwood, 1999; Van Mellaert and Anne´, 2001). Several recombinant DNA technology-based methods are available for Streptomyces spp. (Kieser et al., 2000) and DNA can be introduced into the cell either by protoplastmediated transformation or by conjugation. Screening of aimed transformants might be time-consuming. Traditionally, cultivation of transformants in liquid
* Corresponding author. Tel.: +32-16-337371; fax: +32-16337340. E-mail address:
[email protected] (J. Anne´).
medium was necessary to isolate the requested plasmid DNA to be further analysed (Kieser et al., 2000). More recently, a more advanced method for direct screening of transformants based on PCR was described (Soliveri et al., 1999). However, this protocol remains time-consuming, since it requires several manipulations including collection of mycelium from the plates followed by grinding the mycelium with vortexing in the presence of glass beads, incubation with lysozyme for 30 min and boiling for 15 min prior to PCR. In this report we describe an adaptation of this PCR-based method which allows direct screening of Streptomyces transformants, eliminating the need for extra manipulations before PCR can be carried out, as illustrated here for Streptomyces lividans TK24 and Streptomyces venezuelae ETH14630. During experiments on the analysis of the temperate actinophage VWB (Anne´ et al., 1984) a 1.2- or a 2.0-kb restriction fragment from this temperate phage
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(02)00235-X
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W. Van Dessel et al. / Journal of Microbiological Methods 53 (2003) 401–403
Table 1 Composition of the PCR mixtures used for DNA amplification of Streptomyces transformants for screening purposes Components
Volume (Al) of component for Mix 1
Mix 2
Mix 3
DMSO – cell suspension mix Nuclease-free water Primer A1 (5 AM) Primer A2 (5 AM) 10 SuperTaq polymerase buffer DNTP mix (5 mM each) SuperTaq DNA polymerase (0.5U/Al)
5 27 5 5 5 2.5 0.5
2.5 29.5 5 5 5 2.5 0.5
1.25 30.75 5 5 5 2.5 0.5
Final DMSO concentration (%)
10
5
2.5
was cloned into the widely used Streptomyces multicopy vector pIJ486 (Ward et al., 1986) containing a thiostrepton resistance gene as selection marker. For these cloning experiments, pIJ486 was digested with EcoRI (Invitrogen Life Technologies, Paisley, UK) and subsequently treated with alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) to avoid self-ligation. On the other hand, pGEM-T Easy vector (Promega, Madison, USA) containing the 1.2and 2.0-kb fragments of phage DNA, respectively, was digested with EcoRI. After agarose gel electrophoresis, the obtained 1.2- and 2.0-kb EcoRI fragments were extracted from the gel using the Qiaquick Gel extraction kit (Qiagen, Hilden, Germany). Subsequently, the fragments were ligated in pIJ486 using T4 DNA ligase (Roche Diagnostics), and protoplasts of S. lividans TK24 and S. venezuelae ETH14630 were transformed with these ligation mixtures using a protocol as described (Kieser et al., 2000). After transformation, the mixtures were plated on modified R2 medium (Anne´ et al., 1990) and incubated at 27 jC for 24 h to allow regeneration. Subsequently the plates were overlayed with 1 ml of a thiostrepton (Calbiochem, La Jolla, USA) suspension (500 Ag/ml H2O) (Kieser et al., 2000) and further incubated for 3 to 4 days until the colonies reached a diameter of 2 to 3 mm. A number of colonies were randomly selected to be screened for the presence of the insert. Therefore, the amount of cells of approximately half of each colony was picked up from the plate using a sterile pipette tip and resuspended in 10 Al of a 100% dimethyl sulfoxide (DMSO) solution (Merck, Darmstadt, Germany). Mycelium of S. lividans colonies was difficult to homogenize and remained clumpy, but
this did not hamper efficient PCR. This reaction was carried out with SuperTaq DNA Polymerase (HT Biotechnology, Cambridge, UK) and using primers adjacent to the polylinker site of pIJ486 [A1 ( = 5VCGCAATTCCTTTAGTTGTTCC-3V) and A2 ( = 5VGCGAAACGATCCTCATCC-3V); Soliveri et al., 1999]. The PCR protocol comprised several consecutive steps: 2 min at 95 jC for DNA denaturation, followed by 30 cycles consisting of 30 s at 95 jC, 1 min at 37 jC for annealing and 3 min at 72 jC for elongation, as commonly used for Streptomyces DNA amplifications. To optimise the approach, several reaction conditions were tested using different mixtures. The composition of these mixtures is given in
Fig. 1. Gel electrophoresis of PCR samples on a 1% agarose gel. Lane 1: E-DNA, EcoRI + HindIII digest as a marker; lanes 2 – 4: mycelium-free control; lanes 5 – 7: plasmid-free S. venezuelae control; lanes 8 – 10: S. venezuelae [pIJ486]; lanes 11 – 13: S. venezuelae [pIJ486_1.2-kb insert]; lanes 14 – 16: S. venezuelae [pIJ486_2.0-kb insert]. Each set of three consecutive lanes: mix 1 (10% DMSO), mix 2 (5% DMSO), mix 3 (2.5% DMSO). Lane 17: PCR on plasmid [pIJ486_1.2-kb insert] DNA; lane 18: PCR on plasmid [pIJ486_2.0-kb insert] DNA.
W. Van Dessel et al. / Journal of Microbiological Methods 53 (2003) 401–403
Table 1. PCR-amplified DNA was analyzed by gel electrophoresis using 5 Al of each sample. As negative controls, similar PCRs were performed as for the samples to be analyzed, but either without Streptomyces mycelium in the DMSO solution, or using plasmid-free Streptomyces mycelium. As a positive control, PCR on plasmid DNA isolated from the same transformants was carried out using the previously described protocol. In the latter case, DNA was isolated using an alkaline lysis method (Kieser et al., 2000) after culturing the colonies in 5 ml phage medium (Korn et al., 1978) for 20 to 36 h. The PCR was carried out in a way identical with the one described above with PCR Mix 1 (see Table 1) with the exception that 1 Al of template DNA and 5 Al of a 100% DMSO solution (resulting in a final DMSO concentration of 10%) were used. Fig. 1 shows the results of the improved PCRbased screening for S. venezuelae ETH14630 transformed with empty pIJ486 vector (lanes 8 – 10) or containing a 1.2-kb (lanes 11– 13) or 2.0-kb insert (lanes 14 – 16). The small PCR products (224 bp) (lanes 8 – 10) consist of the polylinker amplified region, since primers A1 and A2 anneal just upstream and downstream of the polylinker site of pIJ486. Amplification reactions with Mixes 1, 2 and 3 (see Table 1) each gave a PCR-fragment of the expected size (Fig. 1). From a comparison of the lanes, it could be concluded that the different concentrations of DMSO used in the amplification reaction did not result in a significant difference in intensity of the fragments on gel. However, no PCR products were obtained if DMSO was omitted or if the amount of DMSO was increased to 20% (results not shown). As expected, in the negative controls (lanes 2 – 7) no PCR products were observed. The obtained results matched well with the positive controls, i.e. plasmid DNA samples prepared according to the protocol previously described (Kieser et al., 2000). In summary, it can be concluded that the here presented adaptation for the preparation of samples to screen Streptomyces transformants by the PCR approach is a major improvement compared to the existing method, since it dramatically reduces the number of manipulations. As a consequence this
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method is far less time-consuming without a loss of specificity and sensitivity.
Acknowledgements This research was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), grant 1.5.1.7.01 from the Fund for Scientific Research in Flanders (FWO) and grant QLK3-2000-00122 from the European Union. W.V.D. and N.G. are IWT research fellows.
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