Letters in Applied Mi(rohioloyy 1991. 13, 21-24
ADONIS 0266825491000742
DNA recovery and direct detection of Tn5 sequences from soil S O N J AS E L E N S K& A W . K L I N G M U L L E RDepartment * Of'Genetics, Uniuersity of Bayreuth, Uniuersitatsstrasse 30, W-8580 Bayreuth, Germanj RG/05: received 18 March 1991 and accepted 19 March 1991 SELENSKA S ., & K L I N G M U L L E RW , . 1991. DNA recovery and direct detection of Tn5 sequences from soil. Letters in Applied Microbiology 13, 21-24. Specific Tn5 sequences inserted in the genome of Enterobacter agylomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed
The prospect of routine release of genetically engineered micro-organisms into the environment requires the development of methods not only for measuring their survival but also for monitoring the fate of their genetic material. Thus the development of methods for direct molecular analysis of soil probes is important. The direct isolation of DNA from soil was pioneered by Ogram et al. (1988). The resulting DNA contains large amounts of brown coloured humic material and is considered too impure for molecular experiments. Steffan et al. (1988) improved the quality of recovered DNA by introducing additional cleaning steps. This increased the complexity of the procedure and caused large losses of DNA. According to the authors the recovery of radioactively labelled DNA was approximately 10%, thus invalidating the method for ecological and risk assessment experiments. We present a simple and effective procedure for preparation of soil DNA, pure enough for molecular analysis.
Materials and Methods B A C T E R I A L STRAIN'S, P L A S M I D S A N D
SOIL I N O C U L A T l O N S
Enterobacter agglomerans 19-1-1 Nay, Km'/ Nm', a strain containing one copy of Tn5
* Corresponding
author
inserted in its n f plasmid (Klingmiiller et ul. 1990; Klingmuller 1991) and also two copies of Tn5 inserted in the chromosomal DNA (this work); and E. agglomerans 339 Sm', a strain cured of its n f plasmid by heat treatment, and free of Tn5, were used. Samples of 50 g sandy loam soil from the experimental field of Bayreuth University were distributed into Erlenmeyer flasks and inoculated with approximately lo7 bacterial cells per g soil, resuspended in Luria broth or saline, as described by Klingmiiller et al. (1990). The flasks were then incubated at 22-C. For a description of E . agglomerans nif' plasmids and the labelling procedure see Singh et al. (1983), and Klingmiiller et al. (1989). A 5 kb HpuI core part of Tn5 was obtained from plasmid pSUP2021 (Simon et a/. 1983) and used as a probe for DNA-DNA hybridization. ISOLATION OF TOTAL SOIL D N A
Two grams wet weight of soil were suspended in 5 ml 0.12 mol/l Na,HPO, (pH 8), containing 1% SDS, were shaken in a water bath at 70°C for 1 h and then centrifuged 15min at 2800 x g at 10°C. The supernatant fluid was stored at 4°C. The DNA was extracted from the pellet two additional times with 6 ml 0.12 mol/l Na,HPO,, pH 8 at 70'C for 20min. The resulting supernatant fluids from all three extractions were collected in one tube at 4°C and centrifuged 30min at 8000 x g. 5 mol/l NaCl (1/10 volume of supernatant fluid) was
22
Sonja Selenska and W . Klingmiiller
added to the clear supernatant fluid and the DNA in it was precipitated with PEG 6OOO (final concentration 15%) overnight. The DNA was then pelleted by centrifugation at 5000 x g and resuspended in 3.7 ml TE buffer (10 mmol/l Tris-HC1, 1 mmol/l EDTA). CsCl (4.1 g) and ethidium bromide (0.5 ml, 5 mg/ml) were added. Such samples had a refraction index of 1.386 and were centrifuged for 16 h at 18OOOO x g in 5 ml tubes in a vTi65 rotor. The DNA band was recovered and dialysed for 2 h against TE buffer at 4°C. Ethidium bromide was extracted with Tris-saturated phenol and the dialysis was continued overnight. The resulting DNA was stored for future analysis at 4°C. Five p1 of each sample were analysed, without any further concentration, in a 0 4 % agarose gel.
a
b
0
3
7
28
42
56 D N A TECHNIQUES
All manipulations with DNA have been performed by standard methods (Sambrook et al. 1989). Electrophoresis of EcoRI digested DNA was performed in 0.8% agarose gel in a TBE buffer system. Southern blotting and hybridization were according to the PhotoGene Instruction manual (BRL-Cat No. 8192SA). Labelling of the 5kb HpaI Tn5 core fragment was as described in BioNick Labeling System Protocol (BRL-Cat. No. 8247SA). Results and Discussion By the procedure developed here we recovered up to 1OOpg DNA from 2 g soil. This is five times more than previously reported (i.e. 1OOOpg per lOOg soil, Steffan et al. 1988; Sayler et al. 1989). The molecular weight of the DNA obtained was about 25 kbp. The A,,,/A,,, ratio varied from 1.6 to 2.0 and the A,,,/A,,, ratio from 1.0 to 1.3. Soil DNA recovered by our simple procedure did not inhibit either the restriction endonucleases tested (EcoRI, PstI, BamHI and HindIII) or Taq polymerase (results not shown). As shown in Fig. 1 it was possible to detect the Tn.5 sequences in total DNA recovered from soil 70 d after inoculation with about lo7 bacterial cells per gram of soil. Viable counts (on Luria-Bertani agar medium, 2 d incubation at 30°C) of the inoculated bacteria showed a strong decrease for the later sampling times, and beyond day 63 viable E. agglomerans 19-1-1 cells expressing the Km and Nm resist-
63
70
-I
-2
Fig. 1. Hybri-slot analysis of total DNA recovered from soil at different days after its inoculation with bacterial cells: (a) 20 pl of DNA recovered from soil samples inoculated with 2.8 x lo7 cells/g soil of Tn5 labelled Enterobacter agglomerans 19-1- 1, resuspended in Luria broth; (b) 20 pI of DNA recovered from soil samples inoculated with 3.6 x lo7 cells/g soil of Tn5 labelled E. agglomerans 19-1-1, resuspended in saline; c-I, 25 pl DNA recovered from noninoculated soil; c-2, 20 pl DNA from soil inoculated with 2.6 x lo7 cells/g soil of wild type E. agglomerans free of Tn5. The 2&25 pl DNA subsamples contained approximately 2 pg of DNA. Abundance of bacteria in inocula was determined by plate counts.
ances coded by Tn5 were no longer detected (limit of detection 10 cells/g soil). We digested with EcoRI endonuclease DNA from soil samples drawn at different days after inoculation and analysed the resulting patterns. In 22 analysed samples including the DNA from day 70 we had positive hybridization reactions of the Tn5 probe with the same bands as in the
Tn5 detection in soil 1
2
3
4
5
6
7
23
soil particles a n d so be protected from degradation - ( Lorenz and Wackernagel 1989). Detected sequences containing Tn5 could also originate from indigenous bacterial recipients that accepted and maintained them. This kind of genetic exchange has been shown to take place in soil (Henschke & Schmidt 1990; Stewart & Singalliano 19901.
Acknowledgements
We thank Mrs M. Steinlein for excellent technical assistance. This work was supported by G r a n t No. 6496-1053-29388 from Bayerisches Staatministerium fur Landesentwicklung und Umweltfragen, Munchen.
References
Fig. 2. Detection of specific EcoRl fragments of Tn5 labelled Enterobacter agglomerans 19-1-1 in soil DNA recovered at different days after soil inoculation. (a) Electrophoresis of EcoRI digested soil DNA recovered: (2) 3 d after inoculation with Tn5 labelled E. agglomerans 19-1-1; (3) 70 d after inoculation with the same bacteria as (2); (4) 3 d after inoculation with Tn5 free E . agglomerans: (5) DNA recovered from noninoculated soil; (1) & (6) total DNA of Tn5 carrying E . agglomerans 19-1-1, digested with EcoR1; (7) kilobase ladder (BRL). (b) Southern blot of (a) and PhotoGene detection of Tn5 containing sequences.
positive control (see Fig. 2), hence the transposon was not moved. All these d a t a were obtained without amplification of DNA by polymerase chain reaction (PCR) used by others in similar experiments (Chaudhry et al. 1989; Bej et al. 1990). W e suggest that the DNA detected a t d a y 70 in o u r soil samples could represent nonculturable inoculated bactera as well as extracellular DNA released from dead bacteria. In recent years d a t a have been presented indicating that some bacteria change into a non-culturable state soon after release into the environment (Byrd & Colwell 1990). Other d a t a show that DNA released from dead organisms can stick t o
BEJ, A.K., STEFFAN,R.J., DICESARE, J., HAFF, L. & ATLAS,R.M. 1990 Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 56, 307-314. BYRD,J.J. & COLWELL, R.R. 1990 Maintenance of plasmid pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment. Applied and Environmental Microbiology 56, 2 104-2107. CHAUDHRY, G.R., TORANZOS, G.A. & BHATTI,A.R. 1989 Novel method for monitoring genetically engineered microorganisms in the environment. Applied and Enoironmental Microbiology 55, 1301-1304. HENSCHKE, R.B. & SCHMIDT,F.R.J. 1990 Plasmid mobilization from genetically engineered bacteria to members of the indigenous soil microflora in situ. Current Microbiology 20, 105-1 10. KLINGM~LLER, W., HERTERICH, S. & MIN, B.W. 1989 Selftransmissible n i j plasmids in Enterobacter. In Nitrogenfixation with Nonlegumes ed. Skinner, F.A. er al. pp 173-178. Kluwer Academic: Dordrecht. KLINGM~LLER, W., DALLY, A,, FENTNER, CH. & STEINLEIN, M. 1990 Plasmid transfer between soil bacteria. In Bacterial Genetics in Natural Environments ed. Fry, J.C. & Day, M.J. pp. 133-151. London: Chapman and Hall. KLINGM~LLER, W. 1991 Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterohacter. FEMS Microbiology Ecology (in press). M.G. & WACKERNAGEL, W. 1989 Adsorption LORENZ, of DNA to sand and variable degradation rates of adsorbed DNA. Applied and Environmental Microbiology, 53, 2945--2952. OGRAM, A., SAYLER, G.S. & BARKAY, T.J. 1988 DNA extraction and purification from sediments. Journal of Microbiological Methods 7, 57-66. J., FRITSCH,E.F. & MANIATIS, T. 1989 SAMBROOK, Molecular Cloning: A Laboratory Manual 2nd edn. New York: Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
24
Sonja Selenska and W . Klingmuller
SAYLER, G.S., FLEMING, J., APPLEGATE, B., WERNER,C. & NIKBAKHT 1989 Microbial community analysis using environmental nucleic acid extracts. In Recent Advances in Microbial Ecology, Proceedings of the 5th International Symposium on Microbial Ecology (ISMES) ed. Tsutomu Harroti et al. pp. 658462. Japan Scientific Societies Press. SIMON,R., PRIEFER,U. & WHLER, A. 1983 A broad host range mobilization system for in uivo genetic engineering: transposon mutagenesis in Gram negative bacteria. BiolTechnology 1, 784-791. SINGH,M., KLEEBERGER, A. & K L I N G ~ L L EW. R ,1983
Location of nitrogen fixation (nif) genes on indigenous plasmids of Enterobacter agglomerans. Molec-
ular and General Genetics 190,373-318. STEFFAN,R.J., COKSOYR, J., BEJ,A.K. & ATLAS,R.M. 1988 Recovery of DNA from soils and sediments. Applied and Environmental Microbiology 54, 29082915. STEWART, G.J. & SINGALLIANO, C.D. 1990 Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic sediments. Applied and Environmental Microbiology 56, 1818-1824.
ADONIS 0266825491000742
DNA recovery and direct detection of Tn5 sequences from soil S O N J AS E L E N S K& A W . K L I N G M U L L E RDepartment * Of'Genetics, Uniuersity of Bayreuth, Uniuersitatsstrasse 30, W-8580 Bayreuth, Germanj RG/05: received 18 March 1991 and accepted 19 March 1991 SELENSKA S ., & K L I N G M U L L E RW , . 1991. DNA recovery and direct detection of Tn5 sequences from soil. Letters in Applied Microbiology 13, 21-24. Specific Tn5 sequences inserted in the genome of Enterobacter agylomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed
The prospect of routine release of genetically engineered micro-organisms into the environment requires the development of methods not only for measuring their survival but also for monitoring the fate of their genetic material. Thus the development of methods for direct molecular analysis of soil probes is important. The direct isolation of DNA from soil was pioneered by Ogram et al. (1988). The resulting DNA contains large amounts of brown coloured humic material and is considered too impure for molecular experiments. Steffan et al. (1988) improved the quality of recovered DNA by introducing additional cleaning steps. This increased the complexity of the procedure and caused large losses of DNA. According to the authors the recovery of radioactively labelled DNA was approximately 10%, thus invalidating the method for ecological and risk assessment experiments. We present a simple and effective procedure for preparation of soil DNA, pure enough for molecular analysis.
Materials and Methods B A C T E R I A L STRAIN'S, P L A S M I D S A N D
SOIL I N O C U L A T l O N S
Enterobacter agglomerans 19-1-1 Nay, Km'/ Nm', a strain containing one copy of Tn5
* Corresponding
author
inserted in its n f plasmid (Klingmiiller et ul. 1990; Klingmuller 1991) and also two copies of Tn5 inserted in the chromosomal DNA (this work); and E. agglomerans 339 Sm', a strain cured of its n f plasmid by heat treatment, and free of Tn5, were used. Samples of 50 g sandy loam soil from the experimental field of Bayreuth University were distributed into Erlenmeyer flasks and inoculated with approximately lo7 bacterial cells per g soil, resuspended in Luria broth or saline, as described by Klingmiiller et al. (1990). The flasks were then incubated at 22-C. For a description of E . agglomerans nif' plasmids and the labelling procedure see Singh et al. (1983), and Klingmiiller et al. (1989). A 5 kb HpuI core part of Tn5 was obtained from plasmid pSUP2021 (Simon et a/. 1983) and used as a probe for DNA-DNA hybridization. ISOLATION OF TOTAL SOIL D N A
Two grams wet weight of soil were suspended in 5 ml 0.12 mol/l Na,HPO, (pH 8), containing 1% SDS, were shaken in a water bath at 70°C for 1 h and then centrifuged 15min at 2800 x g at 10°C. The supernatant fluid was stored at 4°C. The DNA was extracted from the pellet two additional times with 6 ml 0.12 mol/l Na,HPO,, pH 8 at 70'C for 20min. The resulting supernatant fluids from all three extractions were collected in one tube at 4°C and centrifuged 30min at 8000 x g. 5 mol/l NaCl (1/10 volume of supernatant fluid) was
22
Sonja Selenska and W . Klingmiiller
added to the clear supernatant fluid and the DNA in it was precipitated with PEG 6OOO (final concentration 15%) overnight. The DNA was then pelleted by centrifugation at 5000 x g and resuspended in 3.7 ml TE buffer (10 mmol/l Tris-HC1, 1 mmol/l EDTA). CsCl (4.1 g) and ethidium bromide (0.5 ml, 5 mg/ml) were added. Such samples had a refraction index of 1.386 and were centrifuged for 16 h at 18OOOO x g in 5 ml tubes in a vTi65 rotor. The DNA band was recovered and dialysed for 2 h against TE buffer at 4°C. Ethidium bromide was extracted with Tris-saturated phenol and the dialysis was continued overnight. The resulting DNA was stored for future analysis at 4°C. Five p1 of each sample were analysed, without any further concentration, in a 0 4 % agarose gel.
a
b
0
3
7
28
42
56 D N A TECHNIQUES
All manipulations with DNA have been performed by standard methods (Sambrook et al. 1989). Electrophoresis of EcoRI digested DNA was performed in 0.8% agarose gel in a TBE buffer system. Southern blotting and hybridization were according to the PhotoGene Instruction manual (BRL-Cat No. 8192SA). Labelling of the 5kb HpaI Tn5 core fragment was as described in BioNick Labeling System Protocol (BRL-Cat. No. 8247SA). Results and Discussion By the procedure developed here we recovered up to 1OOpg DNA from 2 g soil. This is five times more than previously reported (i.e. 1OOOpg per lOOg soil, Steffan et al. 1988; Sayler et al. 1989). The molecular weight of the DNA obtained was about 25 kbp. The A,,,/A,,, ratio varied from 1.6 to 2.0 and the A,,,/A,,, ratio from 1.0 to 1.3. Soil DNA recovered by our simple procedure did not inhibit either the restriction endonucleases tested (EcoRI, PstI, BamHI and HindIII) or Taq polymerase (results not shown). As shown in Fig. 1 it was possible to detect the Tn.5 sequences in total DNA recovered from soil 70 d after inoculation with about lo7 bacterial cells per gram of soil. Viable counts (on Luria-Bertani agar medium, 2 d incubation at 30°C) of the inoculated bacteria showed a strong decrease for the later sampling times, and beyond day 63 viable E. agglomerans 19-1-1 cells expressing the Km and Nm resist-
63
70
-I
-2
Fig. 1. Hybri-slot analysis of total DNA recovered from soil at different days after its inoculation with bacterial cells: (a) 20 pl of DNA recovered from soil samples inoculated with 2.8 x lo7 cells/g soil of Tn5 labelled Enterobacter agglomerans 19-1- 1, resuspended in Luria broth; (b) 20 pI of DNA recovered from soil samples inoculated with 3.6 x lo7 cells/g soil of Tn5 labelled E. agglomerans 19-1-1, resuspended in saline; c-I, 25 pl DNA recovered from noninoculated soil; c-2, 20 pl DNA from soil inoculated with 2.6 x lo7 cells/g soil of wild type E. agglomerans free of Tn5. The 2&25 pl DNA subsamples contained approximately 2 pg of DNA. Abundance of bacteria in inocula was determined by plate counts.
ances coded by Tn5 were no longer detected (limit of detection 10 cells/g soil). We digested with EcoRI endonuclease DNA from soil samples drawn at different days after inoculation and analysed the resulting patterns. In 22 analysed samples including the DNA from day 70 we had positive hybridization reactions of the Tn5 probe with the same bands as in the
Tn5 detection in soil 1
2
3
4
5
6
7
23
soil particles a n d so be protected from degradation - ( Lorenz and Wackernagel 1989). Detected sequences containing Tn5 could also originate from indigenous bacterial recipients that accepted and maintained them. This kind of genetic exchange has been shown to take place in soil (Henschke & Schmidt 1990; Stewart & Singalliano 19901.
Acknowledgements
We thank Mrs M. Steinlein for excellent technical assistance. This work was supported by G r a n t No. 6496-1053-29388 from Bayerisches Staatministerium fur Landesentwicklung und Umweltfragen, Munchen.
References
Fig. 2. Detection of specific EcoRl fragments of Tn5 labelled Enterobacter agglomerans 19-1-1 in soil DNA recovered at different days after soil inoculation. (a) Electrophoresis of EcoRI digested soil DNA recovered: (2) 3 d after inoculation with Tn5 labelled E. agglomerans 19-1-1; (3) 70 d after inoculation with the same bacteria as (2); (4) 3 d after inoculation with Tn5 free E . agglomerans: (5) DNA recovered from noninoculated soil; (1) & (6) total DNA of Tn5 carrying E . agglomerans 19-1-1, digested with EcoR1; (7) kilobase ladder (BRL). (b) Southern blot of (a) and PhotoGene detection of Tn5 containing sequences.
positive control (see Fig. 2), hence the transposon was not moved. All these d a t a were obtained without amplification of DNA by polymerase chain reaction (PCR) used by others in similar experiments (Chaudhry et al. 1989; Bej et al. 1990). W e suggest that the DNA detected a t d a y 70 in o u r soil samples could represent nonculturable inoculated bactera as well as extracellular DNA released from dead bacteria. In recent years d a t a have been presented indicating that some bacteria change into a non-culturable state soon after release into the environment (Byrd & Colwell 1990). Other d a t a show that DNA released from dead organisms can stick t o
BEJ, A.K., STEFFAN,R.J., DICESARE, J., HAFF, L. & ATLAS,R.M. 1990 Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 56, 307-314. BYRD,J.J. & COLWELL, R.R. 1990 Maintenance of plasmid pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment. Applied and Environmental Microbiology 56, 2 104-2107. CHAUDHRY, G.R., TORANZOS, G.A. & BHATTI,A.R. 1989 Novel method for monitoring genetically engineered microorganisms in the environment. Applied and Enoironmental Microbiology 55, 1301-1304. HENSCHKE, R.B. & SCHMIDT,F.R.J. 1990 Plasmid mobilization from genetically engineered bacteria to members of the indigenous soil microflora in situ. Current Microbiology 20, 105-1 10. KLINGM~LLER, W., HERTERICH, S. & MIN, B.W. 1989 Selftransmissible n i j plasmids in Enterobacter. In Nitrogenfixation with Nonlegumes ed. Skinner, F.A. er al. pp 173-178. Kluwer Academic: Dordrecht. KLINGM~LLER, W., DALLY, A,, FENTNER, CH. & STEINLEIN, M. 1990 Plasmid transfer between soil bacteria. In Bacterial Genetics in Natural Environments ed. Fry, J.C. & Day, M.J. pp. 133-151. London: Chapman and Hall. KLINGM~LLER, W. 1991 Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterohacter. FEMS Microbiology Ecology (in press). M.G. & WACKERNAGEL, W. 1989 Adsorption LORENZ, of DNA to sand and variable degradation rates of adsorbed DNA. Applied and Environmental Microbiology, 53, 2945--2952. OGRAM, A., SAYLER, G.S. & BARKAY, T.J. 1988 DNA extraction and purification from sediments. Journal of Microbiological Methods 7, 57-66. J., FRITSCH,E.F. & MANIATIS, T. 1989 SAMBROOK, Molecular Cloning: A Laboratory Manual 2nd edn. New York: Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
24
Sonja Selenska and W . Klingmuller
SAYLER, G.S., FLEMING, J., APPLEGATE, B., WERNER,C. & NIKBAKHT 1989 Microbial community analysis using environmental nucleic acid extracts. In Recent Advances in Microbial Ecology, Proceedings of the 5th International Symposium on Microbial Ecology (ISMES) ed. Tsutomu Harroti et al. pp. 658462. Japan Scientific Societies Press. SIMON,R., PRIEFER,U. & WHLER, A. 1983 A broad host range mobilization system for in uivo genetic engineering: transposon mutagenesis in Gram negative bacteria. BiolTechnology 1, 784-791. SINGH,M., KLEEBERGER, A. & K L I N G ~ L L EW. R ,1983
Location of nitrogen fixation (nif) genes on indigenous plasmids of Enterobacter agglomerans. Molec-
ular and General Genetics 190,373-318. STEFFAN,R.J., COKSOYR, J., BEJ,A.K. & ATLAS,R.M. 1988 Recovery of DNA from soils and sediments. Applied and Environmental Microbiology 54, 29082915. STEWART, G.J. & SINGALLIANO, C.D. 1990 Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic sediments. Applied and Environmental Microbiology 56, 1818-1824.
