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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 [DELTA]thiL 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 [DELTA]thiL, 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).

A study was conducted to examine the role of single alanine substitution at the Bacillus subtilis transcription factor SpoOA on the activity of spoIIG promoters. The amino acid substitution in SpoOA which suppressed the phenotype of the H359R substitution in sigmaA subunit of the RNA polymerase showed that H359R substitution on sigmaA prevented its interaction with SpoOA. Results suggested the relevance of the region of SpoOA near position 229 in sigmaA-dependent promoter activation.

The significance of the interaction of Spo0A and the sigma subunit of RNA polymerase in the activation of the Bacillus subtilis spoIIG promoter was analyzed. Results of the study revealed that B. subtilis Spo0A activates form both sigmaA-and-sigmaH dependent promoters. Alanine substitutions at these positions were discovered to be significantly reduced from the sigmaA-dependent, Spo0A-dependent promoters, spoIIG and spoIIE in vivo.

The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.