Fate of Transforming DNA After Uptake by Competent Bacillus subtilis : Failure of Donor DNA to Replicate in a Recombination-Deficient Recipient

Abstract

The fate of radioactively-labeled transforming DNA was studied in a recombination-deficient strain of Bacillus subtilis that carried the recB2 mutation (Rec-) Bacterial transformation is an advantageous system for the study of recombination, since the fate of purified donor DNA can be studied in the course of the different steps before, during, and after integration. Recombinationdeficient mutants of bacteriophage and bacteria have been useful in the study of recombination processes (1-10). Recombination deficient, radiation-sensitive mutants of Bacillus subtilis have been isolated and described by several investigators (11)(12)(13)(14). This paper reports the results of experiments in which the fate of transforming DNA was examined in one of these mutants (recB2). Our results indicate that in recB2, the donor-recipient complex is formed to the same extent as in an isogenic Rec+ strain, but that donor DNA fails to replicate in the recipient after formation of the complex. MATERIALS AND METHODS Bacterial strainsThe bacterial strains used were derivatives of Bacillus subtilis 168. Strains BD170 (thr-5 trp-2) and BD191 (thr-5 trp-2 recB2) were derived from the same parent and were isogenic for all markers except the recB2 marker, which was introduced by transformation as was described (15). The recB2 marker is derived from the mitomycin-sensitive strain mc-1 (12). RecB2 has been shown to be radiation sensitive and deficient in both transformability and transducibility (12)(13)(14). Strain BD204 (hisB2 thy) was used to prepare radioactively-labeled transforming DNA.BD55 (trp-2 hisBW), known also as SB25, was used to assay the biological activity of DNA fractions isolated from the transformed cells. The trp-2 and hisB2 markers are cotransformed at a frequency of about 60%. Media and solutionsThe media for plating and for the development of competence were prepared as described (16). Transformed cells were washed in SP buffer (0.15 M NaCl-0.02 M K2HPO4, pH 7.5). The washed samples were resuspended in lysis medium (0.05 M NaCl-0.1 M EDTA, pH 6.9).SPII medium contained Spizizen salts (17), 0.02% casamino acids, 0.1% Difco yeast extract, 0.5% glucose, 0.5 mM CaC12, 2.5 mM MgCl2, and 50,gg/ml of each required amino acid. Re-extraction experimentsCompetent cells were prepared and frozen as was described (16). Frozen competent Rec+ and Rec-cells were quickly thawed and incubated for 5 min at 370C. To begin the reaction, [3H]DNA, isolated from BD204 as described (16), was then added to a final concentration of 1-2 gg/ml. The specific activity of the DNA preparations ranged from 4 to 6 X 105 cpm/htg. The mixture was incubated at 370C for 7 min or 9 min, at which time an excess (100 ,Ag/ml) of salmonsperm DNA (Calbiochem) was added to stop further DNA uptake. In some experiments the reaction mixture was then transferred to 300C. At different times after addition of the salmon-sperm DNA, samples of the transformed cells were chilled rapidly, washed three times by centrifugation in icecold SP, and resuspended in lysis ...

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

“…Similar observations were made when using a different transformation reporter system ( Figure 3-figure supplement 3 and Video 3 , see below). In line with current models of transformation (Davidoff-Abelson and Dubnau, 1971; Ephrussi-Taylor and Gray, 1966; Fox and ALLEN, 1964; Gabor and Hotchkiss, 1966; LACKS, 1962; Méjean and Claverys, 1984; Piechowska and Fox, 1971), these observations are consistent with a model in which recombination occurs by direct integration of the ssDNA donor and forms a hetero-duplex. Therefore, at least one round of DNA replication and division is required to generate two different homo-duplex chromosomes in progeny cells ( Figure 3F ).…”

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

“…Similar observations were made when using a different transformation reporter system (Figure 3-figure supplement 3 and Video 3, see below). In line with current models of transformation (Davidoff-Abelson and Dubnau, 1971;Ephrussi-Taylor and Gray, 1966;Fox and ALLEN, 1964;Gabor and Hotchkiss, 1966;LACKS, 1962;Méjean and Claverys, 1984;Piechowska and Fox, 1971), these observations are consistent with a model in which recombination occurs by direct integration of the ssDNA donor and forms a hetero-duplex. Therefore, at least one round of DNA replication and division is required to generate two different homo-duplex chromosomes in progeny cells ( Figure 3F).…”

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

“…Bacillus subtilis strains (Table 2) were constructed by standard methods (Davidoff‐Abelson and Dubnau, 1971) from wild‐type strain PY79 (Youngman et al ., 1984). Sporulation was induced by resuspension at 37°C and samples were collected at indicated times ( t n ) after the initiation of sporulation (Sterlini and Mandelstam, 1969).…”