In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this DeltathiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the DeltathiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).