Control of the Synthesis of Macromolecules During Amino Acid and Thymine Starvation in Bacillus subtilis

Abstract

Studies of Maal0e, Lark, and others with amino acidand thymine-starved cultures revealed successive steps in the biosynthesis of Escherichia coli chromosomes. In this study, the corresponding mechanisms in Bacillus subtilis 168 were examined. Using a strain requiring both thymine and tryptophan, we found that, 3 hr after the start of amino acid starvation, when the deoxyribonucleic acid (DNA) content of the culture had increased 40 to 50%, DNA synthesis ceased. After 4 to 5 hr, 100%o of the cells were immune to thymineless death; their chromosomes had presumably been completed. Immune cultures slowly incorporated 3H-thymine. Thymine incorporation increased 20-fold 30 min after readdition of amino acids, indicating reinitiation of chromosome synthesis. Simultaneous presence of amino acids and thymine was required for reinitiation. If 5-bromouracil (5-BU) was added instead of thymine, newly replicated DNA segments could be separated by centrifugation in CsCl. Analysis of the CsCl fractions by a transformation assay showed that the order in which the markers were synthesized was ade-16, thr-5, leu-8, metB5. Less than half the chromosomes started resynthesis synchronously in 5-BU. Nevertheless, chromosome alignment in the amino acid-starved culture is probably very good: marker frequency analysis of its DNA gives the same normalized frequencies as DNA from "perfectly" aligned spores. Full viability is maintained in the chromosome-arrested culture for 10 hr in thymine-free medium in the absence or presence of amino acids. In the latter condition, protein synthesis proceeds, and the cells filament and become more lysozyme-sensitive. Such cells must be incubated and plated on hypertonic or on slow-growth media; otherwise, they undergo "quasiosmotic" thymineless death. This death is thus apparently not directly attributable to any damage of chromosomal DNA. Further, weakening of the teichoic acid portion of the cell wall is not involved, since 32p incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits 32P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue. Preceding experiments have shown that deoxyribonucleic acid (DNA) synthesis proceeds sequentially along the chromosome from a fixed starting point in exponentially growing cells of Escherichia coli (5, 19, 26) and Bacillus subtilis (32). In B. subtilis, this demonstration is based on two types of experiments. First, a comparison by transformation of marker frequencies in DNA from exponential cultures with marker frequencies in DNA of stationary

Cell-wall thickening in Bacillus subtilis. Comparison of thickened and normal walls

“…There was no clear indication of localized wall thickening in the limited number of sections examined, and obviously further work is required including serial sectioning of cells with thickened walls. However, similar provisional results were obtained by Landman and colleagues (Miller et al 1967;Anraku & Landman, 1968) who observed an approximately twofold uniform thickening of walls of B. subtilis incubated in the presence of chloramphenicol. Studies in vivo (Chung, Hawirko & Isaac, 1964;Chung, 1967) have shown that growth in Bacillus cereus and Bacillus megaterium proceeds by the localized addition of new material to the existing wall.…”

“…The reason for this limited synthesis is not known. Anraku & Landman (1968) reported that [32P]Pl incorporation into teichoic acid is strongly inhibited by chloramphenicol. Experimentally their procedures differed greatly from those we have used.…”

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

“…The ARCHER AND LANDMAN same experiment was performed with nonsynchronized cultures. Transformants for fu-r were scored on plates containing, in addition to the requirements of the strain, 1 ,ug of fu per ml. ( Table 1).…”

Development of Competence in Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

“…Our results show clearly that chromosome replication is not essential for the development of competence since, in our cultures, competence first appears and develops long after DNA synthesis has been blocked. There is no net DNA synthesis in cultures of chromosome-arrested B. subtilis (3).…”

“…The procedure used for arrest of chromosomes at the terminus through amino acid starvation was previously published (3).…”