The proposal deals with the heterogeneity in organization of the conserved and redundant rRNA (rm) operons, their expression and the molecular mechanisms involved in the regulation of ribosome synthesis in the endospore-forming bacteria Bacillus subtilis and has three major goals: 1) To study the differential expression and growth rate control of the 10 rrn by creating single-copy lacZ fusion capable of integrating at the native rrn site or at heterologous loci (amyE, thrC). 2) To establish the role and interaction of the tandemly arranged promoters (P1,P2) and Upstream Activating Sequences (UAS) in growth rate regulation by studying the expression of promoter parts and after saturation mutagenesis in vital regions (-10, -35, -50, and -88) of various rrn operons as a function of different growth rates or during amino acid starvation in relA+ and relA- 3) To establish the role of specific rrn, RNA polymerase and the stringent control system (relA) in the life cycle of B. subtilis by inducing deletions or transposon (Tn917) insertions into selected rrn and isolating mutants defective in growth regulation and to ascertain how these mutants function during vegetative growth, sporulation and germination. The experimental approach involves recombinant DNA techniques of plasmid constructions, gene fusions to the E. coli lacZ gene, in vivo and in vitro mutagenesis, primer extension analysis, B-galactosidase measurements and assays of (p)ppGpp accumulation.
