Chromosome Replication in Sporulating Cells of Bacillus subtilis

Author:

Sargent Michael G.1

Affiliation:

1. Division of Microbiology, National Institute for Medical Research, London, NW7 1AA, England

Abstract

A method of specifically labeling the chromosomal terminus of Bacillus subtilis is described. When sporulating cultures were pulse-labeled with [ 3 H]thymidine and then treated with 6-( p -hydroxyphenylazo)uracil, a drug which inhibits deoxyribonucleic acid (DNA) synthesis rapidly and completely, the only labeled spores formed were those that had completed replication during the pulse period. DNA-mediated transformation was used to show that the DNA of spores formed in the presence of 6-( p -hydroxyphenylazo)uracil had the same ratio of origin to terminus markers as DNA from untreated spores. Furthermore, spores formed in the presence of 6-( p -hydroxyphenylazo)uracil had the same DNA content as untreated spores. These two observations indicated that spores formed in the presence of 6-(hydroxyphenylazo)uracil contained completed chromosomes. The rate of termination of chromosomes destined to be packaged into spores was determined by this method, using the Sterlini-Mandelstam replacement system and a single medium exhaustion system for inducing sporulation. With both systems the rate of termination reached a broad peak 2 h after the start of sporogenesis. This was measured from the time of resuspension by using the replacement system and from the point where exponential growth ceased in the exhaustion system. The amount of spore DNA synthesized in the Sterlini-Mandelstam sporulation-inducing medium was very close to one-half the amount of the DNA present in mature spores. This suggests that chromosomes destined to be packaged into spores were replicated from close to the origin and possibly initiated in the sporulation-inducing medium. A method was devised for estimating the time taken to complete replication of the chromosomes destined to be packaged into spores. This was probably no more than 50 min. Whereas starvation must have occurred almost simultaneously in most cells in the population, the chromosome replication that was essential for sporogenesis was distributed over a wide time span. Thus, in some cells, replication started within 10 min of the nutritional step-down, but the peak rate was not reached for 1 h; thereafter replication continued at a substantial rate.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

Reference37 articles.

1. Aubert J.-P. A. Ryter and P. Schaeffer. 1969. Fate of spore DNA during a new spore cycle in Bacillus subtilis p. 148-158. In L. Leon Campbell (ed.) Spores IV. American Society for Microbiology Washington DC.

2. Synthesis of ribosomal ribonucleic acid during sporulation of Bacilus subtilis;Bonamy C.;J. Bacteriol.,1973

3. Inhibition of bacterial DNA replication by 6-p-hydroxy-phenylazouracil. Differential effect on repair and semiconservative DNA synthesis in B. subtilis;Brown N. C.;J. Mol. Biol.,1971

4. Completed chromosomes in thymine-requiring BaciUus subtilis spores;Callister IL;J. Bacteriol.,1974

5. Homogeneity in Bacillus subtilis spore DNA content;Callister H.;J. Mol. Biol.,1976

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