Chloramphenicol Restores Sigma Factor Activity to Sporulating Bacillus subtilis

Abstract

The a subunit of RNA polymerase from sporulating Bacillus subtilis is markedly inhibited in its ability to direct active transcription of phage 4e DNA in vitro. Treatment of sporulating bacteria with chloramphenicol rapidly restores a activity, suggesting that sporulating cells contain an inhibitor ofa that is physiologically unstable or that becomes unstable after drug treatment. The hypothetical inhibitor is depleted exponentially with an apparent half-life of 11 min at 37°.The onset of sporulation by Bacillus subtilis is associated with a marked decrease in the ability of the a subunit of RNA polymerase to direct active transcription of phage Ye DNA in vitro (1, 2). Although sporulating bacteria contain as much a polypeptide as vegetative bacteria, a from sporulating B. subtilis is functionally inhibited and only weakly associated with RNA polymerase (3). It was previously observed that B. subtilis phage ye is unable to grow in sporulating bacteria (4) and that the time course of decrease in phage burst size during the first hours of spore formation closely parallels the decrease in a activity, as indicated by the reduced ability of RNA polymerase to transcribe he DNA in vitro (5). In contrast, a sporulation-defective mutant of B. subtilis that partially retains a activity supports 4e growth during late stationary phase (6).To test the idea that the failure of he to grow in wild-type sporulating cells is related to the inhibition of a activity (5) (Fig. 1), we found that the rates of total RNA synthesis after infection of vegetative and sporulating bacteria were approximately 0.95 and 0.17 nmol/min per 108 cells, respectively (Table 1). Next we determined the fraction of RNA labeled at 2-4 min after infection that hybridized to denatured he DNA ( Fig. 2A). About 22% of the pulse-labeled RNA from infected vegetative cells and about 11% of the radioactive RNA from infected sporulating bacteria annealed to Oe DNA. From the rates of total RNA synthesis, and correcting for the efficiency of hybridization, we calculate that the rate of Be transcription in sporulat- The RNA extracted from the infected and pulse-labeled cells of the experiments of Fig. 2A and B was hybridized to heavy (H) strand DNA of B. subtilis and to denatured be DNA (Fig. 2) and the % input hybridized at near saturating amounts of DNA was determined. The rates of total cellular RNA synthesis were estimated from the initial slopes of the curves of Fig. iD (the data are the average of two independent experiments).

Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis

“…Taken together, these results suggest that an interaction with an anti-s A factor and/or a modi®cation of s A should be considered as a possible mechanism(s). Indeed, an inhibitor that interferes with the activity of vegetative RNA polymerase (Es A ) during sporulation has been reported (Linn et al 1973;Segall et al 1974;. This hypothetical inhibitor might be essential not only for sporulation but also for growth.…”

Bacillus Subtilis RNA Polymerase and its Modification in Sporulating and Phage‐Infected Bacteria

“…As discussed above, chloramphenicol restores apolypeptide activity to sporulating B. subtilis. They also report that this drug stimulates the ability of sporulating bacteria to support transcription of &-DNA in vivo, although drug treatment does not affect the rate of total cellular RNA synthesis (33). These workers also find that stationary-phase cells of an asporogenous mutant (LS-3) that retains apolypeptide activity support &-DNA transcription more actively than wild-type sporulating bacteria (41).…”

“…Tjian and Losick (23) have argued that sporulating cells contain a component that inhibits a-polypeptide activity, probably by interfering with the binding of a-polypeptide to polymerase. This hypothetical component appears to be metabolically unstable, since it is rapidly depleted from sporulating cells treated with chloramphenicol, a drug that blocks protein synthesis (33). T h e inhibitor of a-polypeptide could either R N 4 POL.YhlERZ\E be a protein that turns over rapidly during sporulation or, alternatively, a component of sporulating cells that becomes unstable in the presence of chloramphenicol.…”

“…T h e inhibitor of a-polypeptide could either R N 4 POL.YhlERZ\E be a protein that turns over rapidly during sporulation or, alternatively, a component of sporulating cells that becomes unstable in the presence of chloramphenicol. Segall et al (33) calculate that the inhibitor decays with an apparent half-life of 11 min at 37°C. For comparison, the psubunits of polymerase turn over with a half-life of about 5 hr in sporulating cells (34).…”

“…However, unlike Segall et al (33), these workers did not measure the rate of 4e-RNA synthesis in sporulating bacteria, but, rather, they determined only that a fraction of pulse-labeled RNA corresponds to 4e-RNA sequences without taking into account the decrease in the rate of total RNA synthesis that occurs during spore format ion.…”