1887

Microbiology Society logo

Volume 136, Issue 2

Determination of guanine-plus-cytosine content of bacterial DNA by dual-laser flow cytometry Free

Abstract

A dual-laser flow cytometer was used to analyse different species of bacteria for the molar percentage of guanine-plus-cytosine (% G + C) without the need for DNA extraction or purification. Ethanol-fixed bacterial cells were stained with a combination of DNA-specific fluorochromes, Hoechst 33258 and chromomycin A3, which bind to AT- and GC-rich regions of DNA, respectively. A linear relationship ( = 0·99) was demonstrated between the log of the ratio of chromomycin A3 to Hoechst 33258 fluorescence and the log of the % G + C as determined by thermal denaturation ( ) or buoyant density centrifugation (Bd) methods. Linearity was maintained for all bacterial species tested over the range of 28–67% G + C. A standard curve was constructed using five strains whose % G + C had been determined by other methods. From the equation describing this line, the % G + C values of nine other strains with known DNA base composition, together with the five strains used to construct the curve, were calculated using the chromomycin A3 to Hoechst 33258 ratio and were in agreement with values obtained by , Bd or HPLC. The reproducibility of flow cytometric analysis (mean error 0·7% G + C) compared well with the reproducibility of other methods. Mixtures containing two species were also analysed. Two cell populations could be discerned in mixtures containing two species which differed in base composition by as little as 4% G + C. Dual-laser flow cytometric analysis of stained bacteria is a rapid, simple and accurate method for determining the % G + C of bacterial DNA and can be used to distinguish populations of bacteria with differing % G + C content.

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology 1990
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-136-2-359
1990-02-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/136/2/mic-136-2-359.html?itemId=/content/journal/micro/10.1099/00221287-136-2-359&mimeType=html&fmt=ahah

References

  1. Behr W., Honikel K., Hartmann G. 1969; Interaction of the RNA polymerase inhibitor chromomycin with DNA. European Journal of Biochemistry 9:82–92
     [Google Scholar]
  2. Colwell R. R. 1970; Polyphasic taxonomy of the genus Vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus and related Vibrio species. Journal of Bacteriology 104:410–433
     [Google Scholar]
  3. Comings D. E. 1975; Mechanisms of chromosome banding VIII. Hoechst 33258-DNA interaction. Chromosoma 52:229–243
     [Google Scholar]
  4. Cummins C. S., Johnson J. L. 1971; Taxonomy of the Clostridia: wall composition and DNA homologies in Clostridium butyricum and other butyric acid-producing Clostridia. Journal of General Microbiology 67:33–46
     [Google Scholar]
  5. Van den Engh G. J., Trask B. J., Gray J. W., Langlois R. G., Yu L.-C. 1985; Preparation and bivariate analysis of suspensions of human chromosomes. Cytometry 6:92–100
     [Google Scholar]
  6. Gray J. W., Dean P. N., Fuscoe J. C., Peters D. C., Trask B. J., Van Den Engh G. J., Van Dilla M. A. 1987; High-speed chromosome sorting. Science 238:323–329
     [Google Scholar]
  7. Johnson J. L. 1978; Taxonomy of the bacteroides. I. Deoxyribonucleic acid homologies among Bacteroides fragilis and other saccharo- lytic Bacteroides species. International Journal of Systematic Bacteriology 28:245–256
     [Google Scholar]
  8. Johnson J. L. 1985; Determination of DNA base composition. Methods in Microbiology 18:1–31
     [Google Scholar]
  9. Johnson J. L., Cummins C. S. 1972; Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryne- forms, classical propionibacteria, and strains of Arachnia propionica. . Journal of Bacteriology 109:1047–1066
     [Google Scholar]
  10. Johnson J. L., Francis B. S. 1975; Taxonomy of the Clostridia: ribosomal ribonucleic acid homologies among the species. Journal of General Microbiology 88:229–244
     [Google Scholar]
  11. Johnson J. L., Phelps C. F., Cummins C. S., London J., Gasser F. 1980; Taxonomy of the Lactobacillus acidophilus group. International Journal of Systematic Bacteriology 30:53–68
     [Google Scholar]
  12. Ko C. Y., Johnson J. L., Barnett L. B., Mcnair H. M., Vercellotti J. R. 1977; A sensitive estimation of the percentage of guanine plus cytosine in deoxyribonucleic acid by high performance liquid chromatography. Analytical Biochemistry 80:183–192
     [Google Scholar]
  13. Lalande M., Schreck R. R., Hoffman R., Latt S. A. 1985; Identification of inverted duplicated # 15 chromosomes using bivariate flow cytometric analysis. Cytometry 6:1–6
     [Google Scholar]
  14. Langlois R. G., Carrano A. V., Gray J. W., Vandilla M. A. 1980; Cytochemical studies of metaphase chromosomes by flow cytometry. Chromosoma 77:229–251
     [Google Scholar]
  15. Mandel M. 1966; Deoxyribonucleic acid base composition in the genus Pseudomonas. . Journal of General Microbiology 43:273–292
     [Google Scholar]
  16. Mandel M., Schildkraut C. L., Marmur J. 1968; Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. Methods in Enzymology 12:184–206
     [Google Scholar]
  17. Marmur J., Doty P. 1962; Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. Journal of Molecular Biology 5:109–118
     [Google Scholar]
  18. Mesbah M., Premachandran U., Whitman W. B. 1989; Precise measurement of the G + C content of deoxyribonucleic acid by high- performance liquid chromatography. International Journal of Systematic Bacteriology 39:159–167
     [Google Scholar]
  19. Muller W., Gautier F. 1975; Interactions of heteroaromatic compounds with nucleic acids: A-T-specific non-intercalating DNA ligands. European Journal of Biochemistry 54:385–394
     [Google Scholar]
  20. Schildkraut C. L., Marmur J., Doty P. 1962; Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. Journal of Molecular Biology 4:430–443
     [Google Scholar]
  21. Silvestri L. G., Hill L. R. 1965; Agreement between deoxyribonucleic acid base composition and taxometric classification of gram-positive cocci. Journal of Bacteriology 90:136–140
     [Google Scholar]
  22. Starr M. P., Mandel M. 1969; DNA base composition and taxonomy of phytopathogenic and other enterobacteria. Journal of General Microbiology 56:113–123
     [Google Scholar]
  23. Van Dilla M. A., Langlois R. G., Pinkel D., Yajko D., Hadley W. K. 1983; Bacterial characterization by flow cytometry. Science 220:620–622
     [Google Scholar]
  24. Voss J. G. 1970; Differentiation of two groups of Corynebacterium acnes. . Journal of Bacteriology 101:392–397
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-136-2-359
Loading
Determination of guanine-plus-cytosine content of bacterial DNA by dual-laser flow cytometry
Microbiology 136, 359 (1990); https://doi.org/10.1099/00221287-136-2-359
/content/journal/micro/10.1099/00221287-136-2-359
/content/journal/micro/10.1099/00221287-136-2-359
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error