Chromosome Replication in Sporulating Cells of Bacillus subtilis

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.

Density gradient analysis of DNA replicated during Bacillus subtilis sporulation

“…Experiments with 6-(p-hydroxyphenylazo)uracil (HPUra), a specific inhibitor of DNA synthesis in Bacillus subtilis, indicate that successful sporulation depends on the completion of chromosome replication (7). This is'consistent with evidence which indicates that mature spores contain completed chromosomes (17,20). Although termination of DNA replication under starvation conditions is necessary for sporLilation, slowing down the rate of replication with subinhibitory concentrations of HPUra has no effect on the time of appearance of early sporulation-associated events (26).…”

“…Experiments with bacteria undergoing synchronized DNA replication suggest that there is a critical point, some 15 min after the initiation of replication, at which the starvation stimulus becomes effective (14). More recent work (20) indicates that the amount of spore DNA synthesized in the sporulation-inducing medium is roughly one-half the amount found in t Present address: Cellular and Developmental Biology Department, The Biological Laboratories, Harvard University, Cambridge, MA 02138. the mature spore. This suggests that chromosomes destined for inclusion into spores may have to be replicated from close to the origin.…”

Inhibition of Sporulation in Bacillus subtilis by Bromodeoxyuridine and the Effect on DNA Replication

“…Escape from HYUra inhibition of sporulation HPUra had been used previously to show that successful formation of spores depended on the completion of chromosome replication in the starvation medium (Dunn et al, 1978). Spores contain completed chromosomes (Oishi et al, 1964;Sargent, 1980), and it is assumed that all replicating forks present in chromosomes proceed to termination before or during packaging of the DNA into the spore. Once replication has gone to completion, addition of the DNA synthesis inhibitor no longer affects the capacity of the cells to sporulate.…”

Specific labeling of the Bacillus subtilis chromosome terminus

“…1A). With the SP,8-strain there is a low level of hybridization a Sporulation was induced in all strains except RB1124 by method A (10). Sporulation was induced in RB1124 by method B (10).…”

“…The whole region is probably about 5% of the chromosome (8,12). The specificity of the terminus labeling procedure, described previously (10), can be assessed by determining the degree of homology of this DNA with DNA of phage SP,8 or with 43T, which has an attachment site within the same region (13).…”