CC BY
420
SHORT COMMUNICATION
УДК 579.873.11.083.12
potassium HYDRoxiDE-ETHYLENE DIAMINE TETRAACETIC ACID METHoD
for the rapid preparation of small-scale pcr template dna from
ACTINOBACTERIA Zhibin Sun a, Yan Huang a, Yanzhuo Wang a, Yuguo Zhao b*, Zhongli Cuia*
a Key Laboratory of Environmental Microbiology of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China; b Institute of Soil Sciences, Chinese Academy of Sciences, Nanjing, 210008, People's Republic of China
abstract
Genomic DNA extraction from Gram-positive bacteria is a laborious and time-consuming process. A rapid and convenient method was established to extract genomic DNA from a single colony as a PCR template. KOH-EDTA is used as a lysis buffer to disrupt the cell envelope, releasing genomic DNA, and Tris-HCl (pH = 4) is then added to neutralize the lysate. The lysate can be used directly as a template for PCR amplification. 16S rDNA was successfully amplified from Gram-positive bacteria from the genera of Bacillus, Streptomyces, Micromonospora, Nonomuraea, Microbispora, and Staphylococcus. Amplification of the trpB gene indicated that this method could also be applied to the amplification of functional genes. Compared to colony PCR methods without KOH-EDTA, this method is extremely fast and efficient, and it is applicable to high-throughput PCR amplifications.
Key words: PCR; DNA extraction; KOH-EDTA; Gram-positive bacteria.
Actinobacteria are known for their ability to produce various active secondary metabolites, such as antibiotics and enzyme inhibitors [1, 2]. The isolation of actinomycetes from previously unexplored and underexplored environments has become an important strategy for the discovery of new antibiotics [3-5]. During the process of isolation, a large number of strains will be handled. Molecular techniques to identify ac-tinomycete genera in heterogeneous samples have been hampered by the lack of an efficient and rapid DNA extraction procedure that is amenable to high sample throughput because of the composition and complexity of their cell walls [6]. Cells of Gram-negative bacteria and cells of some Gram-positive bacteria from a colony can be used as a PCR template without the extraction of pure DNA, as bacterial cells are lysed in the first denaturation step of PCR. However, this technique is not applicable for all bacteria, such as several types of actinomycetes, because the composition and complexity of actinomycete cell walls vary considerably between different genera, compared to other Gram-positive bacteria [6]. Therefore, DNA template preparation from such biological samples is essential for PCR. KOH-EDTA is an efficient lysis buffer for use in single cell genomic sequencing [7, 8]. in this paper, we describe the application and optimization of a KOH-EDTA method for PCR template preparation directly from an actinomycete colony. We also tested the application of this method in releasing genomic DNA from other Gram-positive bacteria, such as Bacillus en-dospores and Staphylococcus aureus.
The following actinomycetes were used in this research: Streptomyces sp. M13, Streptomyces sp. O33, Streptomyces sp. R30, Streptomyces sp. W1, Streptomyces sp. O59, Streptomyces sp. R19, Streptomyces sp. S59, Streptomyces sp. S62, Micromonospora sp. C13, Nonomuraea sp. P6, and Microbispora sp. H54. These strains were isolated from air-dried soil samples collected from different parts of China. In addition, Bacillus subtilis 168, Bacillus amyloliquefaciens, Staphylococcus aureus and Escherichia coli kept in our laboratory were also used in this study. The 16S rDNA primers were P1 (27F, forward) 5'-AGAGTTTGATCCTGGCTCAG-3' and P2 (1492R, reverse) 5'-TACGGYTACCTTGTTACGACTT-3'
[9]. Amplification of bacterial 16S rDNA fragments was performed in 50 ¿l reaction mixtures, containing 5 ¿l 25 mM Mg-Cl2, 5 ¿l 10x reaction buffer, 4 ¿l 2.5 mM dNTP, 100 pmol of each primer, 2.5 U of Taq DNA polymerase, 10% DMSO and 1-3 ¿l bacterial DNA template prepared by the KOH-EDTA method. dH2O was added to a final volume of 50 ¿l. A negative control was also performed using the same volume of dH2O in place of a DNA template sample. The PCR reaction proceeded in the following steps: pre-denaturation at 95°C for 5 min, followed by 30 cycles of 94°C denaturation 30 s, annealing at 52°C for 30 s, and extension at 72°C for 1.5 min; and a final extension at 72°C for 10 min then performed. trpB gene fragments were amplified in 50 ^l reaction mixtures with primers trpBF 5'-GCGCGAGGACCTGAACAACACCGGCTCAC-ACAAGATCAACA-3' and trpBR 5' -TCGATGGCCGG-GATGATGCCCTCGGTG- CGCGACAGCAGGC-3' [10]. PCR mixtures content for amplifying trpB gene are same with that for bacterial 16S rDNA fragments amplification except that trpBF and trpBR used as primers. The PCR cycling conditions were similar to the 16S rDNA amplification, except annealing at 66°C and extension at 72°C for 1 min. All PCR products were analyzed by electrophoresis in 0.75-1.2% agarose in 1x TAE at a constant voltage of 100 V for 30-40 min.
During KOH-EDTA DNA preparation procedure, a single bacterial colony was transferred to a sterile microcentrifuge tube containing 27 ¿l Tris (10 mM)-EDTA (1 mM) (pH 7.6) with a sterile tooth-pick; then, 3^ 0.4 M K0H-10 mM EDTA was added to the tube and incubated at 70°C for 5 min. Next, 3 ¿l Tris-HCl (pH 4.0) was added to the lysate to adjust the pH of the lysate. The lysate was used directly as a DNA template for PCR amplification. The composition of the lysis buffer, the lysis time and the temperature were optimized. From Fig.
IA, we can clearly observe that bacterial genomic DNA could be effectively released from the aerial mycelia of these actino-mycetes, The DNA concentration was high enough to be visualized on agarose gel, and its quality was pure enough to be used as a template for PCR. 16S rDNA of all the strains that were lysed with our method could be readily amplified (Fig.
IB, lanes 5, 7, 9, and 11). This method was also useful for the preparation of template DNA for PCR from endospores of Bacillus (Fig. 1B, lane 1 and 3), Staphylococcus (Fig. 1B, lane 13) and the Gram-negative bacterium E. coli (Fig. 1B, lane 15). In contrast, heat treatment widely used as a colony PCR methods, could not release DNA from endospores (Fig. 1B, lanes 2 and 4) and partially purified actinomycete strains (lane 6 and 8), or it released DNA with poor efficiency (lane 12).
In order to find the optimal genomic DNA extraction condition, the effects of heating time and temperature were examined. Different heating times (1.5 min, 3 min, 5 min, 10 min, 15 min, 20 min) were used at 70°C, and the release of DNA was examined by amplifying the 16S rDNA from these samples. Purified B. subtilis endospores were also selected in the research because of their high resistance to disruption. Heating times longer than 1.5 min (lanes 2 to 7, Fig. 2A) lysed
SHORT COMMUNICATION
10 11 12 13 14 15 16 17 18
2000 bp
1000 bp 750 bp 500 bp 250 bp 100 bp
Fig. 1. Release of bacterial genomic DNA with KOH-EDTA (A) and colony PCR for the amplification of 16S rRNA genes of selected strains (B). A) M: A/Hind III Marker; lanes 1-8 were Streptomyces sp. M13, Streptomyces sp. O33, Streptomyces sp. R30, Streptomyces sp. W1, Nonomuraea sp. P6, Micromonospora sp. C13, Microbispora sp. H54, and Staphylococcus aureus. Lane 9 was the negative control, KOH-EDTA buffer without bacteria. B) M: DL2000 Marker; lanes 1, 3, 5, 7, 9, 11, 13, 15: DNA templates prepared by KOH-EDTA method; lanes 2, 4, 6, 8, 10, 12, 14, 16: mycelia or bacterial colony used directly for amplification ; lanes 1 and 2: the endospores of B. subtilis 168; lanes 3 and 4: the endospores of B. amyloliquefaciens; lanes 5 and 6: Streptomyces sp. M13; lanes 7 and 8: Nonomuraea sp. P6; lanes 9 and 10: Micromonospora sp. C13; lanes 11 and 12: Microbispora sp. H54; lanes 13 and 14: Staphylococcus aureus; lanes 15 and 16: Escherichia coli; lanes 17 and 18: negative control.
the endospores, and a single clear 1.5 kb length product could be observed in each lane. The intensity of the band increased with the extension of heating time, indicating an increase in the amount of DNA released from the samples. Actually, most Gram-positive bacteria lyse more readily for genomic DNA extraction compared to endospores. Between 1 to 3 min of heating treatment was enough to improve PCR results. The extraction temperature was tested in a gradient (25°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C). The endospores of B. subtilis 168, the spores of Nonomuraea sp. P6 and Streptomyces sp. M13 were used. The maximum amplification was obtained at 60 to 70°C (Fig. 2B, lanes 4-6 and lanes 12-14, Fig. 2 C). This is only a temperature range, and the exact temperature may depend on the different strains characteristics. These results also indicate that a higher temperature provides better disruption, (Fig. 2 B, lanes 7-8 and lanes 15-16). KOH-EDTA could disrupt spores of ac-tinomycetes at room temperature (Fig. 2 B, lane 9), but not endospores of bacillus (Fig. 2 B, lane 1).
Besides, we also investigated the function of TE in the KOH-EDTA method. TE was used to suspend the bacterial cells or mycelia in the first step of this method. When TE (pH 7.6) was substituted with Tris-HCl (pH 7.6), the extraction efficiency decreased significantly. Little or no
M1 234 5678
M 1 23 4 56 7 8M9 10 11 121314151617
2000 bp
1000 bp 750 bp 500 bp
2000 bp
1000 bp 750 bp 500 bp 250 bp 100 bp
2000 bp
1000 bp 750 bp 500 bp
250 bp 100 bp
2000 bp
1000 bp 750 bp 500 bp 250 bp
M 1 2 3 4 5 6 7 8 9 M 10 11 12 1314 15 16
Fig. 2. Conditions affecting the release of genomic DNAs by KOH-EDTA method as revealed by 16S rDNA amplification. M: DL2000 Marker; A) Effect of heating time on the release of DNA from the endospores of B. subtilis 168. lane 1: without KOH-EDTA treatment; lanes 2--7: treated with the KOH-EDTA by 1.5, 3, 5, 10, 15, and 20 min, respectively; lane 8: negative control. B) Effects of heating temperature release of DNA from endospores of B. subtilis 168 and Nonomuraea sp. P6. lanes 1 and 9: without KOH-EDTA treatment; lanes 2-8 and lanes 10-16: treated by KOH-EDTA method at different extraction temperatures (lanes 2, 10: 25°C; 3, 11: 50°C; 4, 12: 60°C; 5, 13: 65°C; 6, 14: 70°C; 7, 15: 75°C; 8, 16: 80°C); Lane 17: negative control. C) Different heating temperatures with the KOH-EDTA method for 16S rDNA
amplification from the spores of Streptomyces sp. M13. lane 1: without KOH-EDTA treatment; lanes 2-9: treated with the KOH-EDTA method at different extraction temperatures (lane 2: 25°C, 3: 50°C, 4: 55°C, 5: 60°C, 6: 65°C, 7: 70°C, 8: 75°C, 9: 80°C); lanes 10-11: negative
control. D) 16S rDNA PCR amplification with DNA extracted with the KOH-EDTA method with TE or Tris-HCl. lanes 1-7: DNA samples were obtained by KOH-EDTA method with TE (lane 1: endospores of B. subtilis; 2: endospores of B. amyloliquefaciens; 3: Streptomyces sp. M13; 4: Streptomyces sp. O33; 5: Streptomyces sp. R30; 6: Streptomyces sp. W1; 7: Nonomuraea sp. P6). Lanes 8-9: negative control; lanes
10-16: DNA samples were obtained by the KOH-EDTA method with TE substituted for Tris-HCl (lane 10: endospores of B. subtilis; 11: endospores of B. amyloliquefaciens; 12: Streptomyces sp. M13; 13: Streptomyces sp. O33; 14: Streptomyces sp. R30; 15: Streptomyces sp.
W1; 16: Nonomuraea sp. P6).
М 1 23 456 789 10 11 М
Fig. 3. Colony PCR amplification of the trpB with DNA templates prepared by KOH-EDTA from selected actinomycete strains. M: DL2000 marker; lane 1: Streptomyces sp. M13; lane 2: Streptomyces sp. O33; lane 3: Streptomyces sp. R30; lane 4: Streptomyces sp. W1; lane 5: Streptomyces sp. O59; lane 6: Nonomuraea sp. P6; lane 7: Micromonospora sp. C13; lane 8: Streptomyces sp. R19; lane 9: Streptomyces sp. S59; lane 10: Streptomyces sp. S62; lane 11: Microbispora sp. H54.
DNA could be released from bacillus endospores and actino-mycetes spores by this modified KOH-EDTA method (Fig. 2D, lanes 10-16). These results indicated that TE was essential in the bacterial DNA extraction with KOH-EDTA method. EDTA concentration higher than 1 mM is sufficient for cell lysis.
We further tested the applications of DNA extracted with KOH-EDTA, using it as a template for the amplification of other genes. trpB, a housekeeping gene involved in tryptophan biosynthesis, has often been used as an intra-generic taxonomic marker for Streptomyces. We attempted to amplify the trpB gene by colony PCR from template DNA that was obtained by the KOH-EDTA method. Finally, a single clear amplification band (approximately 800bp) from samples with template from Streptomyces strains could be visualized by agarose gel electrophoresis (Fig. 3).
Rapid DNA preparation is essential for the high throughput genotyping of actinomycete strains in large scale resource collection. Boiling is a very simple DNA extraction method for many microorganisms, but it is not valid for recalcitrant bacteria such actinomycetes. In the colony PCR methods reported for actinomycetes [6, 11], mycelium type and cultivation time were definitive for DNA preparation. Ishikawa [11] developed colony PCR with DNA from mycelia released by heating. According to the protocol, microscopic amounts of mycelia should be used to amplify target DNA fragments, and too many mycelia could cause contamination and inhibit PCR amplification. Though the non-ionic surfactant Nonidet P40 was reported to promote the disruption of the actinomycete cell wall in colony PCR [6], cultures older than 7 days were difficult to disrupt for template DNA amplification. Because the growth rates of different types of actinomycetes are obviously inconsistent, and most members of Streptomyces seem to grow remarkably faster than other rare actinomy-cetes, the application of this surfactant-based colony PCR for high throughput detection is limited. Bead beating with Chelex (BB+C) [12] is a perfect combination of physical and chemical lysis for fast DNA extraction. However, the bead beating method requires a bead beater to treat the samples, and it takes a large amount of time to pre-treat a large number of samples for genomic DNA extraction. Cell disruption with Triton X-100 plus boiling [13] is efficient for DNA extraction from colonies of Lactobacilli. But this method is not effective for Gram-positive enterococci, yielding poor lysis and inconsistent amplifications [14].
KOH-based alkaline lysis is a high-throughput small-scale DNA preparation method. The procedure is simple and does not require special reagents, but appropriate EDTA concentra-
tion is essential for enhancing the effectiveness of DNA extraction. More than 200 strains of bacteria (mostly actinomy-cetes) were treated by this extraction method to obtain template DNA for PCR amplification of 16S rDNA. Among these strains, only 9 bacterial DNA samples extracted by KOH-EDTA method failed to serve as PCR amplification templates, and this failure was not attributed to the extraction method because we can observe genomic DNA bands from the bacterial extraction samples by agarose gel electrophoresis. For these strains, 16S rRNA genes were successfully amplified with DNA obtained by ultrasonication (data not shown). The KOH-EDTA method can effectively shorten the DNA extraction procedure, and it is applicable to multiple types of bacteria with great reproducibility and lower cost.
Special attention should be paid to guarantee highly efficient bacterial genome extraction, we recommend that the lysis reagents (TE, KOH-EDTA, Tris-HCl) be replaced every two months. In addition, the extraction reagents should not be frequently sterilized at a high-temperature. Aliquoting after the first sterilization of the reagents or bacterial filter sterilization may be a judicious choice to ensure the high efficiency of bacterial genome DNA extraction.
ACKNOWLEDGMENT This work is financially supported by grants from Natural Science Foundation of Jiangsu Province, China (No. BK2012029), the Natural Science Foundation of China (31270095), the National Science and Technology Support Program (2012BAD14B02), and the Ministry of Science and Technology of PRC (No. 2008FY110600).
REFERENCES
1. Berdy J. Bioactive microbial metabolites. J Antibiot. 2005; 58: 1-26.
2. LamK.S. Discovery of novel metabolites from marine actinomycetes. Curr. Opin. Microbiol. 2006; 9: 245-51.
3. Jensen P.R., Mincer T.J., Williams P.G., Fenical W. Marine actinomycete diversity and natural product discovery. Anton. Leeuw. Int. J. G. 2005; 87: 43-8.
4. BullA.T., Stach J.E. Marine actinobacteria: new opportunities for natural product search and discovery. Trends Microbiol. 2007; 15: 491-9.
5. Okoro C.K., Brown R., Jones A.L., Andrews B.A., Asenjo J.A., Goodfellow M., and Bull A.T. Diversity of culturable actinomycetes in hyper-arid soils of the Atacama Desert, Chile. Anton. Leeuw. Int. J. G. 2009; 95: 121-133.
6. Gathogo E.W.N., Waugh A.C.W., Peric N., Redpath M.B, Long P.F. Colony PCR amplification of actinomyces DNA. J Antibiot. 2003: 56; 423-4.
7. RaghunathanA., FergusonH.R., Bornarth C.J., Song W., DriscollM., Lasken R.S. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 2005; 71: 3342-7.
8. Rodrigue S., Malmstrom R.R., Berlin A.M., Birren B.W., Henn M.R., Chisholm S.W. Whole genome amplification and De novo assembly of single bacterial cells. PLoS One. 2011; 4: e6864.
9. Frank, J.A., Reich, C.I., Sharma, S., Weisbaum, J.S., Wilson, B.A., Olsen, G.J. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ. Microbiol. 2008; 74: 2461.
10. Guo Y.P., Huang Y. Design and validation of primers for housekeeping genes of streptomycetes. Acta. Microbiol. Sinica. 2007: 47: 1080-3.
11. Ishikawa J., Tsuchizaki N., Yoshida M., Ishiyama D., Hotta K. Colony PCR for detection of specific DNA sequences in actinomyces. Actinomycetologica. 2000; 14: 1-5.
12. Jaffe R.I, Lane J.D, Albury S.V, NiemeyerD.M. Rapid extraction from and direct identification in clinical samples of methicillin-resistant Staphylococci using the PCR. J. Clin. Microbiol. 2000; 38: 3407.
13. Veyrat A., Miralles M.C., Perez-Martinez G. A fast method for monitoring the colonization rate of lactobacilli in a meat model system. J. Appl. Microbiol. 1999; 87: 49-61.
14. Simmon K.E., Steadman D.D., Durkin S., Baldwin A., Jeffrey W.H, Sheridan P., Horton R. Shields M.S. Autoclave method for rapid preparation of bacterial PCR-template DNA. J. Microbiol. Methods. 2003; 56: 143-9.
