A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.The growth rate of a bacterial culture depends on the composition of the growth medium; bacteria are able to adjust their rate of deoxyribonucleic acid (DNA) synthesis to the growth rate. This adjustment is obtained through a change in the frequency of initiation of the replication cycles (9,20,24,25). Thus, the control of the initiation of replication appears to play a fundamental role in the regulation of DNA synthesis, although the features of this control are still not clearly understood.The use of mutants altered in the process of initiation of replication at high temperature should allow a better understanding of the regulatory mechanisms concerned. Many laboratories have obtained mutants in which DNA synthesis is blocked at elevated temperature and some of these organisms appear to carry mutations affecting the initiation process (3,7,11,12,18). This paper describes the properties of a mutant of Bacillus subtilis that is temperature sensitive for the initiation of replication.MATERIALS AND METHODS Bacterial strains. The parental strain from which the Ts37 mutant was derived is W168 Thy-, Trp-, Ura-. 474 The mutant strain W168 Leu-, Met-, Ade-(Mu 8u 5u 16) was used as a recipient in the transformation experiments.Isolation of mutant Ts37. Spores of the parental strain were exposed to ultraviolet irradiation and plated on nutrient agar (Difco). After 2 days of incubation at 30 C, the colonies were replicaplated on the same medium and incubated overnight at 45 C. Colonies present on the 30 C master plate, but absent on the 45 C replica plate, were picked and streaked for further testing.Growth media. The bacteria were cultivated either on nutrient broth (Difco) medium (21) or on semisynthetic medium (minimal Spizizen medium [22] supplemented with 0.5% glucose and 0.02% casein hydrolyzate). The doubling times of the mutant at 30 C on these media are approximately 45 and 60 min, respectively.The two media were supplemented with thymine (5 ;&g/ml), tryptophane (40 ;&g/ml and uracil (20 jg/ ml).Growth of the bacteria. The bacteria were cultivated...
A mutant of Bacillus subtilis unable to initiate a new round of replication at 45 C has been described. Here we show that inhibition of DNA synthesis in this mutant is reversible and that DNA synthesis is resumed at low temperature, even in the presence of chloramphenicol. Initiation of a new replication cycle thus can occur in the absence of protein synthesis. A thermolabile component required for initiation therefore appears to be synthesized at 45 C in an inactive form and can be activated at 30 C in the presence of an inhibitor of protein synthesis. Although resistant to chloramphenicol, the reinitiation of replication occurring after lowering the temperature is sensitive to rifampin and streptolydigin.on July 16, 2020 by guest
We used a Bacillus subtilis mutant described previously, which is temperature sensitive for initiation of replication. The inhibition of deoxyribonucleic acid synthesis occurring at 45 C was shown to be reversed when the temperature is lowered even in the absence of protein synthesis. If the bacteria are returned to 30 C, after a prior period at 45 C, they are able to initiate the first round of replication in the presence of chloramphenicol, but the initiation of the second round still requires protein synthesis. This paper shows that the proteins necessary to initiate the second round of replication can be present in bacteria long before this round is initiated. In addition, the appearance of these proteins seems to be influenced by the length of the previous 45 C period. Although similar reinitiation kinetics are observed at 30 C after prior 45 C periods of 30 or 65 min, the ability to initiate the second round without further protein synthesis appears much earlier after a longer exposure at 45 C. To explain these results, a hypothesis is presented which assumes that two different proteins are both necessary for initiation.
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