Characteristics of a Bacillus subtilis W23 mutant temperature sensitive for initiation of chromosome replication

Abstract

A temperature-sensitive mutant of Bacillus subtilis W23, dna-20 (Ts), has been isolated and shown to be defective in initiation of rounds of chromosome replication at the nonpermissive temperature. Upon transfer of dna-20(Ts) from 30 to 45 C, deoxyribonucleic acid synthesis, as measured by [3H]thymine incorporation, gradually ceases. The distribution of genetic markers among unreplicated and replicated deoxyribonucleic acid, isolated from dna-20(Ts) after a period at 43 C in a medium containing 5-bromouracil, and fractionated in a CsCl gradient, shows that the cessation of initiation at the higher temperature is immediate. On the other hand, ribonucleic acid and protein synthesis continues at elevated or unaltered rates for some time after the shift to 45 C. Marker frequency analysis shows that all rounds of replication in progress at the time of the temperature shift terminate rapidly (within 40 min), even when chromosomes are replicating dichotomously in rich media. dna-20(Ts) remains 100% viable for at least 2 h at 45 C. Over a 5-h period at 45 C the nuclear bodies remain compact; a small number (less than 5%) of deoxyribonucleic acid-less cells are produced, but there is no morphological distortion of the cells. When the cells are returned to 30 C after 2 h at 45 C, chromosome replication is initiated rapidly at the normal origin and then proceeds in the normal established sequence. However, a second round of replication is initiated soon after the first. dna-20(Ts) has been shown to map as a B-group mutation, the major class of initiation mutants identified in B. subtillus 168.

Characterization and mapping of temperature-sensitive division initiation mutations of Bacillus subtilis

“…The initial rise in cell number of tsl indicates that in this mutant partial septa were at first completed, so becoming visible by phase-contrast microscopy, whereas the rise in bacillus number indicates that cell separation was occurring. The subsequent fall in cell number after 30 min at 490C was unlikely to be due to septal resorption, since this would require resorption of septa after their completion. In fact, this fall, which was accompanied by a slight fall in bacillus number, could be accounted for by the considerable cell lysis seen in this mutant at the later times of this experiment.…”

“…Extraction of spore DNA with thioglycolic acid and urea was also as described previously (4), except that extracts were treated with 50 jig of RNase (prepared by the method of Marmur [18]) per ml before the addition of detergent and deproteinized with phenol rather than chloroform-isoamyl alcohol. When vegetative cell DNA was required, it was extracted from cultures in mid-exponential growth and deproteinized with phenol as described previously (30,32). In these cases, care was taken to ensure that both the mutant and its parent strain were growing at the same rate so that the distribution of genetic markers within each strain was as similar as possible.…”

Cell division of cycle of Bacillus subtilis: evidence of variability in period D

“…To account for the dependence of the average cell mass upon the growth rate, one must assume that either the mass at initiation of a round or C + D, C, and/or D vary as a function ofthe growth rate. Reports from several laboratories indicate that C is a constant at growth rates higher than 1.0 doubling per h at 370C and is equivalent to about 40 min (10,22,25,26,27,35,38,39). In two different laboratories the D period has also been found to be constant, independent of the growth rate.…”

Initiation and termination of chromosome replication at 45 degree C in a temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134

“…However, not all of them have been characterized to the same extent. Thus, it has been shown by density transfer experiments in conjunction with measurements of origin and terminus genetic marker activities that dna-1 and Ts37, derivatives of strain 168, and dna-20(Ts), a derivative of strain W23, behave in a manner consistent both with rapid inhibition of initiation of replication at the nonpermissive temperature and with progress of the existing rounds of replication to termination (4,9,11). Similar tests on the other mutants have not been made.…”

“…At mid-exponential growth, the culture was diluted into an equal volume of the same medium at 45°C containing 5-bromouracil (200 .g/ml). After 110 min at 45°C, the culture was processed and fractionated in an equilibrium density gradient of CsCl as described previously (9). Figure 1 shows the distribution of DNA Table 1 for details of recipient strains used in the transformation assays.…”