Uptake of “Single-stranded” DNA in Hemophilus influenzae and its ability to transform

doi:10.1016/S0022-2836(66)80175-4

Journal of Molecular Biology

Volume 16, Issue 2, April 1966, Pages 317-327

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Conditions for the “reversible” and “irreversible” uptake of denatured DNA of Hemophilus influenzae by receptor cells have been described. At a pH of 4·8, cells bound “irreversibly” twice as much denatured DNA as they could bind native DNA under optimal conditions. Spheroplasts, produced by the penicillin technique, and cell wall material, prepared according to the procedure of Hancock & Park (1958), also bound denatured DNA “irreversibly” at low pH. The denatured DNA, bound “irreversibly” to cells, did not penetrate the cells at low pH; however, these cells transformed with relatively low efficiency when returned to neutral pH. The efficiency of transformation of cells that had taken up DNA at pH 4·8 was greatly increased by subsequent treatment with EDTA at pH 7. Fractions from equilibrium density centrifugation provided direct physical proof in support of the idea that the transformations were produced by fully denatured DNA.

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Cited by (19)

Natural transformation of Pseudomonas stutzeri by single-stranded DNA requires type IV pili, competence state and comA
2002, FEMS Microbiology Letters
Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX⁺) or by heat-denatured chromosomal DNA. Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA). Uptake of ³H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA. The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand. Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.
Gene transfer among bacteria in natural environments
1997, Advances in Applied Microbiology
Nucleic acid transport driven by ion gradient across cell membrane
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Mechanism of Bacterial Transformation and Transfection
1974, Progress in Nucleic Acid Research and Molecular Biology
This chapter discusses that in spite of the large amount of information on the physiology of the competent cell, including the production of competence factors in a number of transformation systems and an additional protein that must be synthesized to permit irreversible binding of DNA by D. pneurnoniae, the mechanism of DNA uptake remains obscure. This process requires energy, divalent cations, and results in breakage of incoming DNA. The DNA apparently enters lengthwise, and in the case of one phage DNA, a particular end enters first. The chapter describes that the enzymology of recombination in transformation and transfection is a difficult problem to solve, as there are indications of alternative enzymatic pathways for recombination, so that mutants blocked in one pathway may not necessarily show loss of ability to be transformed or transfected. There is also the unpleasant possibility that cells with deficiencies in more than one pathway may be inviable, making it difficult to assign biological roles to particular enzymes. The chapter discusses that the enzymological studies of transformation and transfection systems, together with information on intermediates and by-products of recombination and the involvement of membranes in recombination, will provide a coherent picture of the mechanism of transformation and transfection during the next few years.
Differential denaturation of Haemophilus influenzae DNA
1971, BBA Section Nucleic Acids And Protein Synthesis
The objective of this study is to determine whether differential denaturation of various genes can be observed. By treatments with heat or by titration with hydroxyl ions at subcritical levels, genetic markers from Haemophilus influenzae have been inactivated in the order of streptomycin resistance > erythromycin resistance > novobiocin resistance. The kinetics and extent of inactivation depends on whether a thermal or alkaline method is used. Results obtained with an assay system which allows the expression of single-stranded DNA are complementary to those testing the transforming activity of double-stranded DNA.
Heterologous DNA nuclear uptake by mouse DNA fibroblasts in vitro and its early fate
1970, Experimental Cell Research
An analysis has been made of some of the factors influencing the uptake of ³H-labeled Escherichia coli deoxyribonucleic acid (DNA) by mice fibroblasts (NCTC cells) in vitro: DNA concentration, incubation time, pH and temperature of the medium, sonication and denaturation of the DNA influence the quantitative uptake of ³H-labelled material. This uptake is reduced by metabolic inhibitors (2,4,dinitrophenol, cyanide, arseniate). The DNA penetrates into the cell nucleus, and a CsCl gradient centrifugation analysis suggests that exogenous DNA penetrates into the cells without being degraded, retaining its double-stranded form during the first step of penetration. On subsequent incubation, the disappearance of tritiated DNA of E. coli characterized by its density corresponds to a labelling increase into cellular DNA.

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Journal of Molecular Biology

Uptake of “Single-stranded” DNA in Hemophilus influenzae and its ability to transform

Biochim. biophys. Acta

J. Mol. Biol.

J. Mol. Biol.

Biochim. biophys. Acta

Biochim. biophys. Acta

J. Mol. Biol.

J. Exp. Med.

J. Exp. Med.

J. Bact.

Proc. Nat. Acad. Sci., Wash.