Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis

Abstract

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage oe. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with the DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phases (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.Among prokaryotic developmental phenomena, the process of spore germination and outgrowth is particularly interesting to study since it is naturally synchronous and characterized by sequential changes in cell structure and gene expression (7). To study the control of gene expression during outgrowth in a simplified system, we have taken advantage of the finding that infection of sporulating cells of Bacillus subtilis by the virulent bacteriophage oe results not only in lack of phage reproduction but also in trapping of the phage genome within the developing spore (23). The resultant carrier spores can produce phage only during outgrowth. An analogous system has been described for Bacillus cereus (5).Control at the level of ribonucleic acid (RNA) synthesis during outgrowth is suggested by the finding of Armstrong et al. (1, 2) that transcription of the B. subtilis chromosome is ordered in time. They observed the synthesis of 16S and 23S ribosomal RNA before RNA of heterogeneous size (taken to be messenger RNA) began to be synthesized. In addition, others have shown that RNA synthesis is required for successful outgrowth (11), that different RNA species are found in outgrowing spores than in either vegetative or sporulating cells (8), that the appearance of certain bacterial proteins is ordered (9,24,28), and that deoxyribonucleic acid (DNA) replication is delayed during outgrowth (12,20,25,29). As a result, we have placed particular emphasis on comparing nucleic acid synthesis in germinating and outgrowing normal and carrier spores in the hope of finding when the carried phage genome begins to be expressed.Since Oe-induces little or no detectable synthesis of phage-specific proteins or DNA in infected sporulating ...

The sigma factors of Bacillus subtilis.

“…However, the difficulty with which E-A is isolated from sporulating cells (25,182) and the loss of bacteriophage gene expression (25,185) following the onset of sporulation suggest that once the early sporulation genes are expressed, A may become largely inactive. If this is so, the possible synthesis of A late in sporulation (242) could represent a device to provide the spore with A that is inactivated and stored for use during germination and outgrowth (33).…”

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis

“…Phage infection of Bacillus has been used to explore the control mechanisms associated with the switchover from vegetative growth to sporulation (6, 9,17) and from dormancy to active growth (3,5). Such studies have indicated that, relative to the capacity for phage production during vegetative growth, phage production at other stages in the cell's life cycle is greatly diminished.…”

Bacillus subtilis mutant temperature sensitive in the synthesis of ribonucleic acid

“…Also, the finding that the RNA synthesis during the early stages of germination is not temperature sensitive in the mutant (Fig. 7) could be explained if, under these conditions, the synthesis of RNA does not depend on sigma factor (5). The effect of the rna-53 mutation on phage SPP1 transcription in vivo (Fig.…”

“…The block of transcription at the restrictive temperature is suppressed by a protein synthesis inhibitor such as chloramphenicol (Fig. [3][4][5], indicating that a metabolically unstable inhibitor might be responsible for the observed effect. The possibility that such an inhibitor is a protease seems very unlikely in view of the experiment shown in Fig.…”

“…It is generally accepted that, in bacteria, the ribonucleic acid (RNA) polymerase determines strategies of gene expression either by interacting with other proteins or through a modification of its own structure; the enzyme-mediated regulations become particularly evident under certain physiological conditions or during phage infection. Accordingly, changes in the pattern of transcription have been shown to occur during sporulation and germination in Bacillus subtilis (2,5,17,22,30,31,34). Early reports of modification of one of the subunits of RNA polymerase during sporulation ofB.…”