Independence of Bacillus subtilis spore outgrowth from DNA synthesis. (1/24)

The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidoxic acid and 6-(p-hydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhbition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors.  (+info)

Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA. (2/24)

In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.  (+info)

Effect of 6-(p-hydroxyphenylazo)-uracil on the homologous and heterologous transduction processes in Bacillus subtilis. (3/24)

We have studied the effect of 6-(p-hydroxyphenylazo)-uracil on the recombination processes that operate in the homologous and heterologous transduction mediated by PBS1 and SP10 phages of Bacillus subtilis. The results obtained demonstrate that the process of heterologous genetic exchange is sensitive to this compound, whereas the homologous process is not. The present data, along with those of our previous work (U. Canosi, A. G. Siccardi, A. Falaschi, and G. Mazza, J. Bacteriol. 126:108--121, 1976), suggest that the DNA polymerase III is involved in the recombination process that operates in transformation and heterologous transduction, whereas homologous transduction follows a partially independent pathway not involving this protein.  (+info)

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant. (4/24)

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.  (+info)

Identification of different sites of expression for spo loci by transformation of Bacillus subtilis. (5/24)

Asporogenous mutants of Bacillus subtilis were rendered capable of forming heat-resistant spores by transformation with wild-type (spo+) DNA at, or near, the start of sporulation. For several mutants up to about 50% of the colonies derived from heat-resistant spores, formed as a result of the transformation, remained genetically asporogenous (spo). This was thought to indicate that the genome of the mother cell, but not that of the forespore, was transformed to spo+ and that correct expression of the spo locus in the mother cell was sufficient for spore formation. At the end of the process the mother cell was destroyed, leaving a mature heat-resistant spore that was genetically asporogenous. It is concluded that the loci spoIIID, spIVA, spoVB and spoVE are expressed in the mother cell. For one mutant more than 99% of the colonies derived from heat-resistant spores were genetically spo+. It is concluded that the locus involved, spoVA, had to be expressed in the forespore. Thus different sporulation-specific loci are expressed in the mother cell and in the forespore. The loci expressed in the mother cell are expressed in one cell type so that another cell type, the forespore, can develop into a heat-resistant spore. Other unselected donor markers could be introduced into the recipient during transformation provided high concentrations of DNA were used. The frequency of congression was the same for spo survivors as for spo+ survivors. This implies that there was no correlation between the DNA strand into which the selected spo+ and the unselected donor markers integrated.  (+info)

Characterization of the global transcriptional responses to different types of DNA damage and disruption of replication in Bacillus subtilis. (6/24)

DNA damage and perturbations in DNA replication can induce global transcriptional responses that can help organisms repair the damage and survive. RecA is known to mediate transcriptional responses to DNA damage in several bacterial species by inactivating the repressor LexA and phage repressors. To gain insight into how Bacillus subtilis responds to various types of DNA damage, we measured the effects of DNA damage and perturbations in replication on mRNA levels by using DNA microarrays. We perturbed replication either directly with p-hydroxyphenylazo-uracil (HPUra), an inhibitor of DNA polymerase, or indirectly with the DNA-damaging reagents mitomycin C (MMC) and UV irradiation. Our results indicate that the transcriptional responses to HPUra, MMC, and UV are only partially overlapping. recA is the major transcriptional regulator under all of the tested conditions, and LexA appears to directly repress the expression of 63 genes in 26 operons, including the 18 operons previously identified as LexA targets. MMC and HPUra treatments caused induction of an integrative and conjugative element (ICEBs1) and resident prophages (PBSX and SPbeta), which affected the expression of many host genes. Consistent with previous results, the induction of these mobile elements required recA. Induction of the phage appeared to require inactivation of LexA. Unrepaired UV damage and treatment with MMC also affected the expression of some of the genes that are controlled by DnaA. Furthermore, MMC treatment caused an increase in origin-proximal gene dosage. Our results indicate that different types of DNA damage have different effects on replication and on the global transcriptional profile.  (+info)

Cloning and characterization of the polC region of Bacillus subtilis. (7/24)

The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme.  (+info)

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication. (8/24)

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.  (+info)