RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase beta subunit gene rpoB causing resistance to rifampin (Rif(r)) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global effects of rpoB mutations on metabolism, wild-type and 11 distinct congenic Rif(r) mutant strains of B. subtilis were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the Rif(r) mutants, including the utilization of novel substrates previously unknown in B. subtilis, such as gentiobiose, beta-methyl-D-glucoside, and D-psicose. The results indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in B. subtilis.