Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

Abstract

Tryptophan-and thymine-requiring cells of Bacillus subtilis, emerging from an amino acid starvation treatment which causes arrest of the chromosomes at the terminus, were not transformable. During subsequent incubation in a thymineless medium supplemented with amino acids, the cultures developed competence while retaining chromosome arrest. The competent subpopulation apparently shares the synchronous chromosome arrest of the bulk population. This was shown by different methods. The principal method was marker frequency analysis of the deoxyribonucleic acid extracted from a population enriched for competent cells by a column-chromatographic method. It is concluded that development of the competent state can occur in nondividing cells, and that the presence of a replication fork actively engaged in synthesis of deoxyribonucleic acid is not required for the development of this state.

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

“…In any case, the effects of selective decrease in transformant number in chromosomearrested cultures were minimized by minimizing the length of incubation in lysozyme prior to plating. Fortunately, the chromosome-arrested cells are exceptionally lysozyme-sensitive (2). At the peak of competence, 10 min of incubation in 250 yg of lysozyme per ml is generally sufficient for complete protoplasting.…”

“…Special procedures were developed to obviate these two difficulties. Chromosome arrest in the competent fraction of our populations was demonstrated by a variety of methods including procedures which used cultures considerably enriched for competent cells (2). In addition, the donor DNA external to the cytoplasmic membrane was made accessible to deoxyribonuclease digestion by lysozyme dissolution of the cell wall at a time when a uniform state of chromosome arrest continued to be maintained.…”

“…Media and procedures. The composition of the media, as well as all procedures used, are described in the preceding paper (2).…”

“…A. in partial fulfillment of the requirements of Georgetown University for the degree of Doctor of Philosophy. 2 Present address: Instituto de Botanica, Universidade do Porto, Porto, Portugal.…”

“…Our approach was as follows. We used the competent chromosome-arrested cultures described in the preceding paper (2) to observe whether such cultures were transformable only for markers at the terminus or whether loci distant from this point could also be transformed. If the transformation frequency in the synchronized recipients for a given marker did not depend on its distance from the terminus, such a result could be explained in two different ways: (i) that the passage of markers through the membrane does not depend on homology and the location of the replication fork, or (ii) that there is such a dependence, but that it is masked, because the DNA for distant markers is held at sites between wall and membrane in the recipient cells and enters only later, when their replication forks are on the move again.…”