Physiological suppression of the temperature-sensitive sporulation defect in a Bacillus subtilis RNA polymerase...

Five hundred putative RNA polymerase mutants of Bacillus subtilis were isolated by selecting for resistance to the RNA polymerase inhibitors rifampin (Rifr), streptovaricin (Strr) or streptolydigan (Stdr). This collection was screened for mutants that were unable to sporulate at the non-permissive temperature of 46 degrees C, yet which sporulated well at 37 degrees C and had normal vegetative growth (Spots phenotype). Nearly one half of the Rifr and one quarter of the Stvr mutants were Spots, whereas none of the Stdr mutants had this phenotype. The streptovaricin resistant strain stv84 was studied in detail. The stv84 mutation maps between cysA14 and strA39 on the B. subtilis chromosome, and the Stvr and Spots phenotypes cotransform at a frequency of 100%. The Spots phenotype of stv84 could be physiologically corrected by supplementing the growth medium with inhibitors of RNA synthesis such as rifampin or azauracil, with carbohydrates such as ribose, mannose or glycerol, or with lipids such as Tween 40 or fatty acids native to Bacillus subtilis membranes. A Spots phenotype resembling that of stv84 was produced in wild type B. subtilis by adding cerulenin, an inhibitor of fatty acid biosynthesis, to the growth medium. This cerulenin-induced sporulation defect was reversed by the same treatments that correct the temperature-sensitive genetic defect of stv84. These data indicate that the Spots phenotype of strain stv84 is not due to an intrinsic inability of the mutant RNA polymerase to transcribe developmentally-specific genes at the nonpermissive temperature. Rather, the data suggest that the stv84 lesion causes a physiological imbalance which disrupts membrane structure or function in sporulating cells.