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

Abstract

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.

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

“…This may be due to inhibitory action of EDTA against DNase which degrade nuclear membrane-bound DNA. The results resemble those reported in Pneumococci genetic transformation (12).…”

“…Glucose in the incubation mixture did not stimulate DNA binding and uptake as had been observed with Pneumococci (12). ATP substantially enhanced the DNA binding and uptake.…”

“…Carbon sources are known to stimulate DNA uptake and transformation in microorganisms (12). In the present soybean nuclei system adding glucose to the incubation medium had no apparent effect on DNA binding and uptake (Table III).…”

“…In genetic transformation experiments with Pneumococci cells, trypsin treatment inhibited the DNA binding to specific proteins (12).…”

LiCl treatment releases a nickase implicated in genetic transformation of Streptococcus pneumoniae

“…The bound DNA undergoes singlestrand breakage. Single-strand breaks similarly formed during binding of DNA to transformable cells in the presence of EDTA (13). Thus, there appears to be a factor on the cell surface which introduces single-strand breaks into doublestranded DNA during the uptake of transforming DNA.…”