The Stability of Messenger Kibonucleic Acid during Sporulation in Bacillus subtiZis*

The stability of postlogarithmic mRNA has been assessed in Bacillus subtilis during sporulation time periods. The appearance of refractile bodies, alkaline phosphatase synthesis, and total protein synthesis is controlled by mRNA which has an estimated half-life of less than 10 min. Inhibition of RNA synthesis at various times by rifampin addition or temperature shift with a temperature-sensitive RNA polymerase mutant demonstrates that postlogarithmic mRNA Is unstable and that continuous RNA synthesis is necessary for expression of the developmental sequence. The addition of chloramphenicol also inhibits the appearance of refractile bodies and alkaline phosphatase synthesis, suggesting that these processes require de nouo protein synthesis. Organism and Growth Jfedium-The parent strain used in all studies was B. subtilis W168 obtained from r\;. Sueoka, Princeton TJniversity. Cells were grown in a modified Schaeffer’s medium containing nutrient’ broth (Difco), 16 g per liter; hIgS04.7H&, 0.5 g per liter; KCl, 2.0 g per liter; 10e3 M Ca(N03)2, lo+ JI MnQ 10eG M FeSOe, and 0.1%; glucose. This medium is designated as 2 x SG. Growth Conditions-St,rains were maintained as colonies on tryptose blood agar base (Difco) plates. For sporulation experiments one colony was inoculated into 20 ml of 2 x SG and incubated with shaking for 6 hours at 37” (late exponential phase). A lo-ml aliquot of this culture was transferred to 200 ml of fresh 2 x SG contained in a 2-liter Erlenmeyer flask. The culture was aerated vigorously at 37” with a rotary shaker. Growth of the culture was followed with a Klett-Summerson calorimeter (No. 66 filter). Three-hundred Klett units are equal to approximately 1 x 109 cells per ml. The percentage of refractile bodies was estimated by courlting 500 cells with a phase contrast microscope. The st,ability of messenger RNA transcribed during Bacillus subtilis sporulation has several implications with respect to the relative importance of transcriptional and translational controIs during the developmental sequence. Conflicting evidence has been obtained supporting the existence of stable (1) and unstable (2, 3) sporulation mRNA in B. subtilis. Until recently the only available inhibitor of RNA synthesis in B. subtilis was actinomycin D. Actinomycin D is known to have undesirable secondary effects unrelated to inhibition of RNA synthesis (4-8). The discovery of a selective inhibitor (rifampin) for DNA-dependent RNA polymerase (9, 10) now allows a rigorous examination of mRNA stability. To confirm the existence of stable mRNA one must demonstrate that sporulation-associated events occur in the presence of a sufficient concentration of rifampin to inhibit greater t,han 95y0 of all RNA synthesis, and that protein synthesis continues at a significant rate during rifampin inhibition of RNA synthesis. These criteria have been satisfied in other differentiating systems (11, 12) but have not been stringently applied to B. subtilis. Preparation of Cell-free E&acts-Cells harvested at, the desired periods of growth were centrifuged (7,000 x g for 6 min), washed in 0.05 M MgCls, 0.01 M Tris-HCI buffer containing 200 pg per ml of sodium glutamate (pH 8.0), and were frozen overnight at -20”. The cells were resuspended in 0.01 M MgC12, 0.2 RI Tris-HCl buffer (pH 8.0). Cells were sonically disrupted at a dial setting of 60 with a Bronwill III sonic oscillator (BP-lll12T probe) at -10”. One-, 2-, 3-, and 4-hour cells (2 ml) were disrupted for 4 min; 5-, 6-, 7-, and S-hour cells (2 ml) were disrupted for 5 min; and 9and lo-hour cells (2 ml) were disrupted for 6 min. Extracts w-ere centrifuged at 17,000 x g for 15 min (4”). Soluble protein was determined by the method of Lowry et al. (13). Alkaline Phosphatase Assay-Alkaline phosphatase (EC 3.1.3.1, orthophosphoric monoester phosphohydrolase) act,ivit,y was determined spectrophotometrically at 410 nm by the method of Loomis (14) except that 2.5 x 10m3 M p-nitrophenyl phosphate was present instead of 1 x 1OW M p-nitrophenyl phosphate and the reaction was run at pH 8.0. In this report we present a number of experiments utilizing rifampin and a temperature-sensitive RNA polymerase mutant which assess the stability of postlogarithmic mRNA in B. subt&s.