The Kinetics of DNA Uptake by Haemophilus influenzae

Stuy, Johan H.; Stern, Doris N.

doi:10.1099/00221287-35-3-391

Cited by 39 publications

(17 citation statements)

References 11 publications

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“…Our basic strategy has been to find a selective inhibitor or experimental condition that would allow normal DNA binding to the cells, but would prevent the nuclease-resistant association between molecules and cells and would also prevent genetic transformation. Published observations suggested that the appearance of nuclease-resistant form of DNA was an energy requiring process (1,5,(7)(8)(9)(10). Table 1 shows that, indeed, several inhibitors of cellular energy metabolism cause a greater decrease in the nuclease-resistant fraction of cell-associated DNA than in the total amount of DNA bound.…”

Section: Resultsmentioning

confidence: 99%

Early Stages in DNA Binding and Uptake During Genetic Transformation of Pneumococci

Seto¹,

Tomasz²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Ethylenediaminetetraacetate and other divalent-cation-complexing agents greatly stimulate the cellular binding of DNA molecules to competent pneumococci, while the appearance of genetic transformants and nuclease-resistant DNA binding are completely inhibited. Based on this finding, we developed an experimental system in which three early and consecutive stages of genetic transformation can be experimentally separated: (i) attachment of DNA molecules to cell surface sites that are only demonstrable in the competent state; (ii) a divalentcation-dependent nucleolytic splitting and release of the adsorbed molecules to the medium; and (iii) emergence of potential transformants accompanied by an energy-requiring and divalent-cation-dependent process in which the cell-associated DNA molecules become inaccessible to shearing forces, nucleases, anti-DNA serum, and polycations.Genetic transformation of pneumococci requires that the recipient cells be "competent" to bind and absorb extracellular DNA molecules. The molecular basis of this capacity seems to involve a subtle alteration of the cell surface, and by am propriate experiments it has been possible to identify several specific macromolecular factors that take part in this surface modification (1). While the sequential appearance of these factors seems clearly associated with the development of competence, it is not known at which stage and for what functions of the transformation process these factors are required.Such an assignment of functions is hampered by the fact that the stages of the transformation process are not well separated in time: attachment of DNA molecules is rapidly followed by an unknown number of subsequent stages. In this communication we describe an experimental system that allows a clear, temporal separation of three consecutive early phases of genetic transformation. This was made possible by our finding that EDTA (and other divalent cation complexing agents) can selectively inhibit stages 2 and 3 of genetic transformation, while allowing DNA molecules to accumulate, attached to binding sites on the cell surface. Our report fully confirms and further develops the observations made by Morrison and Guild (2) and by Lacks (3) concerning the involvement of surface-located nucleases in transformation.

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Section: Resultsmentioning

confidence: 99%

Early Stages in DNA Binding and Uptake During Genetic Transformation of Pneumococci

Seto¹,

Tomasz²

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…Uptake of DNA by other transformable bacteria also requires energy (16)(17)(18), but it is not known whether the energy is required for the initial binding reaction. Donor DNA is also converted to single strands (7,8) and external fragments (3) in B. subtilis, so its entry mechanism may also involve DNase action.…”

Section: Discussionmentioning

confidence: 99%

“…Donor DNA is also converted to single strands (7,8) and external fragments (3) in B. subtilis, so its entry mechanism may also involve DNase action. Haemophilus influenzae presents a different pattern: there is a rapid, energy-dependent uptake of DNA into a DNase-resist-ant form (18), but this DNA remains double stranded (19,20). At a much slower rate donor DNA fragments appear outside the cell (19) and single-stranded segments are inserted into recipient DNA (20).…”

Section: Discussionmentioning

confidence: 99%

Role of a Deoxyribonuclease in the Genetic Transformation of Diplococcus pneumoniae

Lacks

Greenberg

Neuberger

1974

Proc. Natl. Acad. Sci. U.S.A.

106

134

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Two steps in the uptake of DNA by Diplococcus pneumoniae were characterized by analyzing mutants defective in transformation. A strain deficient in the two major deoxyribonucleases of D. pneumoniae takes up DNA normally and converts it to single strands within the cell and oligonucleotide fragments outside the cell. Extracts of this strain contain a residual deoxyribonuclease that produces similar oligonucleotide fragments in vitro. This enzyme is missing in transformationdefective mutants blocked in the second or entry step. Cells of this mutant class bind large amounts of DNA to their surface in a form accessible to external agents.Another class of nontransformable mutants fails to bind DNA at all. Their deoxyribonuclease content is unchanged, and they are apparently blocked in the first or binding step of DNA uptake. The binding step requires a source of energy and prior activation of the cells by competence factor. Entry may be independent of these requirements and may come about by action of the deoxyribonuclease on one strand of DNA with energy for the transport of the intact strand deriving from hydrolysis of the degraded strand. The enzyme may thus act as a DNA translocase.An early step in the genetic transformation of bacteria is the entry of donor DNA into the recipient cell. Prior to entry, DNA appears to be bound to the cell surface in a form sensitive to external agents (1-4). During entry, double-stranded donor DNA is converted to single strands in both Diplococcus pneumoniae (5, 6) and Bacillus subtilii (7,8). The concomitant appearance of donor DNA degradation products in pneumococcal cells suggested the role of a cellular DNase in the entry process (5). Degradation products also appear outside the cells, in proportion to the amount of DNA taken up (3, 9, 10).In a double mutant of D. pneumoniae, which is deficient in the two major DNase activities of the wild type, uptake of DNA and its fate after entry were normal (11). We set out to fractionate and characterize the residual DNase components in this strain, in particular to compare the products of the residual enzymes with the products formed external -to the cell during DNA uptake. We selected mutants of the strain that were more deficient in DNase and, also, mutants that were defective in transformation. Both kinds of mutant were examined with respect to DNase composition, genetic transformability, DNA uptake, and binding of DNA to the outside of the cell. MATERIALS AND METHODSBactrial Strains and Media. Strain designations do. not necessarily indicate genotype. R6endlexo2 and trtlhex4 both are end-i,exo-2 (9, 11). Moendlexo2 and T6trtlhex4 are maltose-negative derivatives. Moendlexo2hex3 is a hex mutant (11). Transformation-defective mutants were obtained in a parental strain after treatment with 1-methyl-3-nitro-1-nitrosoguanidine, and the mutations were transferred by transformation to another strain. A DNase plate assay (11) was used to select noz mutants that failed to form a colorless zone in agar containing DNA and methyl green ...

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“…or washing ( 11). 3 The early stages .of transformation (12,13,14,15) are kinetically similar to phage adsorption (16). That is, the interaction between DNA and a recipient cell results from random collisions, followed by reversible binding of the DNA to the cell, and irreversible binding.…”

Section: Recovery Ofmentioning

confidence: 99%

The effect of osmotic shocking upon Bacillus subtilis transformation

Pierson¹,

Weppner²,

Leach³

1972

Can. J. Microbiol.

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Osmotic shocking of competent Bacillus subtilis reduced transformability by 80–90% through depressing the uptake of radioactive DNA. The supernatant solution from competent cultures restored both transformation and DNA uptake. A factor, which was inactivated by proteolytic enzymes and which was released into the supernatant solution concomitant with the development of maximum competence, was responsible for restoration of transformability. This factor was both heat and pH labile and was produced only by competent strains. While the shock fluid did not restore transformability to shocked ceils, it increased competence 20-fold in a low-transforming strain. The supernatant solution from competent cultures also increased transformability of the low-competence strain. Thus it appears that at least two factors are involved in competence restoration.

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The Kinetics of DNA Uptake by Haemophilus influenzae

Cited by 39 publications

References 11 publications

Early Stages in DNA Binding and Uptake During Genetic Transformation of Pneumococci

Early Stages in DNA Binding and Uptake During Genetic Transformation of Pneumococci

Role of a Deoxyribonuclease in the Genetic Transformation of Diplococcus pneumoniae

The effect of osmotic shocking upon Bacillus subtilis transformation

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