Isolation and Characterization of New Thiamine-Deregulated Mutants of Bacillus subtilis

Abstract

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 ⌬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 ⌬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).

Both Thiamine Uptake and Biosynthesis of Thiamine Precursors Are Required for Intracellular Replication ofListeria monocytogenes

“…In agreement with other groups, we propose the name "ThiT" for Lmo1429 and its homologs such as YuaJ of B. subtilis (8,31). Growth in the presence of pyrithiamine.…”

“…(for example Bacillus weihenstephanensis: 43% identity and 65% similarity over a length of 185 aa and E value of 10 Ϫ38 ) and also those of Streptococcus spp., Lactobacillus spp., or Enterococcus faecalis. The genome of L. monocytogenes does not contain genes with sequence similarities to the presumably thiamine precursor-specific ABC transporter encoded by the ykoFEDC (thiUVWX) operon of B. subtilis (31). To provide experimental evidence for the thiamine transport activity of Lmo1429, we constructed the deletion mutant EGD⌬thiT.…”

A Novel Class of Modular Transporters for Vitamins in Prokaryotes

“…The YkoEDC system was predicted to be involved in salvaging the thiamine precursor hydroxymethylpyrimidine, based on the positional clustering of the ykoEDC cassette with the thiaminase II gene tenA, and some other thiamine salvage genes (34). Genetic and microarray studies of B. subtilis mutants in the thiT and ykoD genes demonstrated that both mutations result in a derepression of thiamine-regulated genes, confirming the involvement of thiT and ykoEDC in the uptake of thiamine and its precursors (38). The thiamine-related transporter ThiW was predicted to be involved in the uptake of the thiazole precursor of thiamine based on the colocalization of the thiW with thiM genes, with the latter encoding the thiazole salvage enzyme hydroxyethylthiazole kinase (34).…”

Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

“…Membrane proteins of unknown function whose genes are associated with the TPP riboswitch have subsequently been shown to be involved with TPP and thiamin transport (Rodionov et al 2002;Winkler et al 2002;Schyns et al 2005), illustrating how characterization of RNA genetic control elements can lead toward description of protein function. Likewise, knowledge of the function of a protein encoded by an mRNA can aid in establishing the ligand identity of an associated riboswitch.…”