Summary
Competent Streptococcus sanguis treated with non-lethal doses of coumermycin Al immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity. The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.
Similar content being viewed by others
References
Alberts, B., Sternalanz, R.: Recent excitement in the DNA replication problem. Nature 269, 655–661 (1977)
Bauer, W., Vinograd, J.: The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J. Mol. Biol. 33, 141–171 (1968)
Bauer, W., Vinograd, J.: Interaction of closed circular DNA with intercalative dyes. II. The free energy of superhelix formation in SV40 DNA. J. Mol. Biol. 47, 419–435 (1970)
Biswas, G.D., Ravin, A.W.: Heterospecific transformation in Pneumococcus and Streptococcus. IV. Variation in hybrid DNA produced by recombination. Mol. Gen. Genet. 110, 1–22 (1971)
Champoux, J.J.: Proteins that affect DNA conformation. Annu. Rev. Biochem. 47, 449–479 (1978)
Crawford, L.V., Waring, M.J.: Supercoiling of polyoma virus DNA measured by its interaction with ethidium bromide. J. Mol. Biol. 25, 23–30 (1967)
Cummings, D.J.: Sedimentation and biological properties of T-phages of Escherichia coli. Virology 23, 408–418 (1964)
Deddish, P.A., Ravin, A.W.: Relation of macromolecular synthesis in Streptococci to efficiency of transformation by markers of homospecific and heterospecific origin. J. Bacteriol. 117, 1158–1170 (1974)
Delius, H., Worcel, A.: Electron microscopic studies on the folded chromosomes of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 38, 53–58 (1973)
Depew, R.E., Wang, J.C.: Conformational fluctuations of DNA helix. Proc. Natl. Acad. Sci. U.S.A. 72, 4275–4279 (1975)
Drilca, K., Snyder, M.: Superhelical Escherichia coli DNA: Relaxation by coumermycin. J. Mol. Biol. 120, 145–154 (1978)
Dubnau, D., Cirigliano, C.: Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: Nonrequirement of deoxyribonucleic acid replication for uptake and integration of transforming deoxyribonucleic acid. J. Bacteriol. 113, 1512–1514 (1973)
Gellert, M., Mizuuchi, K., O'Dea, M.H., Nash, H.A.: DNA gyrase: An enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci. U.S.A. 73, 3872–3876 (1976a)
Gellert, M., O'Dea, H.M., Itoh, T., Tomizawa, J.: Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. U.S.A. 73, 4474–4478 (1976b)
Gellert, M., Mizuuchi, K., O'Dea, M.H., Itoh, T., Tomizawa, J.: Nalidixic acid resistance: A second genetic character involved in DNA gyrase activity. Proc. Natl. Acad. Sci. U.S.A. 74, 4472–4476 (1977)
Griffith, J.D.: Visualization of procaryotic DNA in a regularly condensed chromatin like fiber. Proc. Natl. Acad. Sci. U.S.A. 73, 563–567 (1976)
Holloman, W.K., Wiegand, R., Hoessli, C., Radding, C.M.: Uptake of homologous single-stranded fragments by superhelical DNA: A possible mechanism for initiation of genetic recombination. Proc. Natl. Acad. Sci. U.S.A. 72, 2394–2398 (1975)
Higgins, P.N., Peebles, C.L., Sugino, A., Cozzarelli, N.R.: Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzyme activity. Proc. Natl. Acad. Sci. U.S.A. 75, 1773–1777 (1978)
Marians, K.J., Ikeda, J., Schlagman, S., Hurwitz, J.: Role of DNA gyrase in ØX replicative-form replication in vitro. Proc. Natl. Acad. Sci. U.S.A. 74, 1965–1968 (1977)
McEwen, C.R.: Tables of estimating sedimentation through linear concentration gradients of sucrose solution. Anal. Biochem. 20, 114–149 (1967)
Mizuuchi, K., Nash, H.A.: Restriction assay for integrative recombination of bacteriophage λ DNA in vitro: Requirement for closed circular DNA substrate. Proc. Natl. Acad. Sci. U.S.A. 73, 3524–3528 (1976)
Mizuuchi, K., Gellert, M., Nash, H.A.: Involvement of supertwisted DNA in integrative recombination of bacteriophage lambda. J. Mol. Biol. 121, 375–392 (1978)
Pulleyblank, D.E., Shure, M., Tang, D., Vinograd, J., Vosberg, H.: Action of nicking-closing enzyme on supercoiled and nonsupercoiled circular DNA: Formation of a Boltzman distribution of topological isomers. Proc. Natl. Acad. Sci. U.S.A. 72, 4280–4284 (1975)
Radding, C.M.: Genetic recombination: Strand transfer and mismatch repair. Annu. Rev. Biochem. 47, 847–880 (1978)
Raina, J.L., Ravin, A.W.: Enhanced transformability with heterospecific deoxyribonucleic acid in a Streptococcus sanguis mutant impaired in ribonucleic acid polymerase activity. J. Bacteriol. 127, 380–391 (1976)
Raina, J.L., Metzer, E., Ravin, A.W.: Fate of heterospecific transforming DNA bound to Streptococcus sanguis. J. Bacteriol. 133, 1223–1231 (1978)
Raina, J.L., Metzer, E., Ravin, A.W.: Translocation of the presynaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide. Mol. Gen. Genet. 170, 249–259 (1979)
Raina, J.L., Ravin, A.W.: Fate of homospecific transforming DNA bound to Streptococcus sanguis. J. Bacteriol. 133, 1212–1223 (1978)
Ravin, A.W.: A quantitative study of autogenic and allogenic transformations in Pneumococcus. Exp. Cell Res. 7, 58–82 (1954)
Ravin, A.W.: Linked mutations borne by deoxyribonucleic acid controlling the synthesis of capsular polysaccharide in Pneumococcus. Genetics 45, 1387–1404 (1960)
Ravin, A.W., De Sa, J.D.H.: Genetic linkage of mutational sites affecting similar characters in Pneumococcus and Streptococcus. J. Bacteriol. 87, 86–96 (1964)
Ravin, A.W., Chakrabarti, T.: Genetic hybridization at the unlinked thy and str loci of Streptococcus. Genetics 81, 223–241 (1975)
Ryan, M.J.: Coumermycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli: I. In vivo characterization. Biochemistry 15, 3769–3777 (1967)
Ryan, M.J., Wells, R.D.: Coumermycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. II. In vitro characterization. Biochemistry 15, 3778–3782 (1976)
Silverstein, E., Mehta, B.M.: Fate of Bacillus subtilis transforming deoxyribonucleic acid incorporated into transformable Diplococcus pneumoniae. Biochemistry 10, 783–691 (1971)
Stonington, O.G., Pettijohn, D.E.: The folded chromosome of Escherichia coli isolated in protein-DNA-RNA-complex. Proc. Natl. Acad. Sci. U.S.A. 68, 6–9 (1971)
Sugino, A., Peebles, C.L., Kreuzer, K.N., Cozzarelli, N.R.: Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relation to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. U.S.A. 74, 4767–4771 (1977)
Tiraby, J.G., Fox, M.S.: Marker discrimination in transformation and mutation of Pneumococcus. Proc. Natl. Acad. Sci. U.S.A. 70, 3541–3545 (1973)
Vinograd, J., Lebowitz, J., Watson, R.: Early and late helix-coil transition of circular DNA: The number of superhelical turns in polyoma DNA. J. Mol. Biol. 33, 193–197 (1968)
Wang, J.C.: Interaction between DNA and Escherichia coli protein ω J. Mol. Biol. 55, 523–533 (1971)
Worcel, A., Burgi, E.: On the structure of folded chromosome of Escherichia coli. J. Mol. Biol. 71, 127–147 (1972)
Author information
Authors and Affiliations
Additional information
Communicated by M.M. Green
Rights and permissions
About this article
Cite this article
Raina, J.L., Ravin, A.W. Superhelical DNA in Streptococcus sanguis: Role in recombination in vivo. Molec. Gen. Genet. 176, 171–181 (1979). https://doi.org/10.1007/BF00273211
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00273211