A MUTANT CYSTEINYL-TRANSFER-RNA SYNTHETASE AFFECTING TIMING OF CHROMOSOMAL REPLICATION INITIATION IN BACILLUS-SUBTILIS AND CONFERRING RESISTANCE TO A PROTEIN-KINASE-C INHIBITOR

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degr ees C to the protein kinase C (PKC) inhibitor, sphinganine, and temper ature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperatur e, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a ch ange in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a n ovel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a prot ein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins no rmally involved in controlling chromosomal replication initiation in B .subtilis.