Fate of heterologous deoxyribonucleic acid in Bacillus subtilis

M Piechowska, A Soltyk, D Shugar
1975 Journal of Bacteriology  
CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thytrp-. Shortly after uptake, most of the radioactive E. coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor
more » ... DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with E. coli 2H and 3Hlabeled DNA. Association of radioactivity with recipient DNA decreased to onefourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the E. coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication. In some earlier studies it was reported that the fate of heterologous deoxyribonucleic acid (DNA) taken up by competent bacterial cells, and examined by means of fractionation in a CsCl gradient, was somewhat similar to that of homologous DNA. This presumed resemblance was based on the identity of the secondary structures of heterologous and homologous molecules after entry into the recipient cells, and on the fact that, in some instances, the label of heterologous nontransforming DNA was incorporated into recipient DNA like that of homologous transforming DNA. However, the experimental data did not distinguish between incorporation of heterologous DNA label as mononucleotides resulting from intracellular enzymatic degradation or as large fragments via recombination, as in the case of transforming DNA (3, 13, 19, 25) . Although generally accepted that recombination requires similarities in base composition and sequence of the reacting DNA molecules (12), the minimal degree of homology necessary remains to be established. Conse-quently, the absence of transformation by foreign DNA does not, a priori, exclude the possibility of (i) recombination occurring without subsequent expression of the markers introduced, perhaps because of the specificity of the cellular DNA-directed RNA polymerases (15), or (ii) integration of fragments much smaller than those involved in transformation, i.e., so small that, for a limited number of recombination events, the probability of introducing some selected marker is simply that for spontaneous mutation frequencies. The foregoing considerations are of interest in relation to two reported biological effects resulting from the uptake of heterologous nontransforming and nontransfecting DNA by competent bacteria, viz., the lethal effect on streptococci (21) and the mutagenic effect on Bacillus subtilis (17). In neither instance has the molecular mechanism involved been established.
doi:10.1128/jb.122.2.610-622.1975 fatcat:or6hv4y3gjc23d4mnydo7ocqtq