pmc.ncbi.nlm.nih.gov

High diversity in DNA of soil bacteria

Abstract

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

782

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bendich A. J., Anderson R. S. Characterization of families of repeated DNA sequences from four vascular plants. Biochemistry. 1977 Oct 18;16(21):4655–4663. doi: 10.1021/bi00640a020. [DOI] [PubMed] [Google Scholar]
  2. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  3. Britten R. J., Kohne D. E. Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science. 1968 Aug 9;161(3841):529–540. doi: 10.1126/science.161.3841.529. [DOI] [PubMed] [Google Scholar]
  4. Chassy B. M., Giuffrida A. Method for the lysis of Gram-positive, asporogenous bacteria with lysozyme. Appl Environ Microbiol. 1980 Jan;39(1):153–158. doi: 10.1128/aem.39.1.153-158.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crombach W. H. DNA base composition of soil arthrobacters and other coryneforms from cheese and sea fish. Antonie Van Leeuwenhoek. 1972;38(2):105–120. doi: 10.1007/BF02328082. [DOI] [PubMed] [Google Scholar]
  6. DELEY J., VANMUYLEM J. SOME APPLICATIONS OF DEOXYRIBONUCLEIC ACID BASE COMPOSITION IN BACTERIAL TAXONOMY. Antonie Van Leeuwenhoek. 1963;29:344–358. doi: 10.1007/BF02046087. [DOI] [PubMed] [Google Scholar]
  7. De Ley J. Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J Bacteriol. 1970 Mar;101(3):738–754. doi: 10.1128/jb.101.3.738-754.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Ley J., Tijtgat R. Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie Van Leeuwenhoek. 1970;36(4):461–474. doi: 10.1007/BF02069048. [DOI] [PubMed] [Google Scholar]
  9. Dhillon S. S., Rake A. V., Miksche J. P. Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA. Plant Physiol. 1980 Jun;65(6):1121–1127. doi: 10.1104/pp.65.6.1121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Escara J. F., Hutton J. R. Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate. Biopolymers. 1980 Jul;19(7):1315–1327. doi: 10.1002/bip.1980.360190708. [DOI] [PubMed] [Google Scholar]
  11. Fuhrman J. A., Comeau D. E., Hagström A., Chan A. M. Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Appl Environ Microbiol. 1988 Jun;54(6):1426–1429. doi: 10.1128/aem.54.6.1426-1429.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geck P., Nász I. Concentrated, digestible DNA after hydroxylapatite chromatography with cetylpyridinium bromide precipitation. Anal Biochem. 1983 Dec;135(2):264–268. doi: 10.1016/0003-2697(83)90681-4. [DOI] [PubMed] [Google Scholar]
  13. Gillis M., De Ley J., De Cleene M. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur J Biochem. 1970 Jan;12(1):143–153. doi: 10.1111/j.1432-1033.1970.tb00831.x. [DOI] [PubMed] [Google Scholar]
  14. Gillis M., de Ley J. Determination of the molecular complexity of double-stranded phage genome DNA from initial renaturation rates. The effect of DNA base composition. J Mol Biol. 1975 Nov 5;98(3):447–464. doi: 10.1016/s0022-2836(75)80079-9. [DOI] [PubMed] [Google Scholar]
  15. Holben William E., Jansson Janet K., Chelm Barry K., Tiedje James M. DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community. Appl Environ Microbiol. 1988 Mar;54(3):703–711. doi: 10.1128/aem.54.3.703-711.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hutton J. R. Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea. Nucleic Acids Res. 1977 Oct;4(10):3537–3555. doi: 10.1093/nar/4.10.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lindahl T., Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618–3623. doi: 10.1021/bi00769a019. [DOI] [PubMed] [Google Scholar]
  18. Lindahl T., Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610–3618. doi: 10.1021/bi00769a018. [DOI] [PubMed] [Google Scholar]
  19. McConaughy B. L., Laird C. D., McCarthy B. J. Nucleic acid reassociation in formamide. Biochemistry. 1969 Aug;8(8):3289–3295. doi: 10.1021/bi00836a024. [DOI] [PubMed] [Google Scholar]
  20. Rake A. V. Isopropanol preservation of biological samples for subsequent DNA extraction and reassociation studies. Anal Biochem. 1972 Aug;48(2):365–368. doi: 10.1016/0003-2697(72)90088-7. [DOI] [PubMed] [Google Scholar]
  21. Somerville C. C., Knight I. T., Straube W. L., Colwell R. R. Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl Environ Microbiol. 1989 Mar;55(3):548–554. doi: 10.1128/aem.55.3.548-554.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Steffan R. J., Atlas R. M. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl Environ Microbiol. 1988 Sep;54(9):2185–2191. doi: 10.1128/aem.54.9.2185-2191.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Steffan R. J., Goksøyr J., Bej A. K., Atlas R. M. Recovery of DNA from soils and sediments. Appl Environ Microbiol. 1988 Dec;54(12):2908–2915. doi: 10.1128/aem.54.12.2908-2915.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Torsvik V., Salte K., Sørheim R., Goksøyr J. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl Environ Microbiol. 1990 Mar;56(3):776–781. doi: 10.1128/aem.56.3.776-781.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]