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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).

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a [[sigma].sup.A]-type promoter bearing the TG dinucleotide motif of 'extended' -10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified [[sigma].sup.A]-containing RNA polymerase (E[[sigma].sup.A]), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by E[[sigma].sup.A] during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

The DNA binding protein GerE stimulates transcription from several promoters that are employed by RNA polymerase containing sigmaK during endospore formation in Bacillus subtilis. The model that GerE interacts with sigmaK at the cotX promoter was tested by looking for amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE. Findings showed that amino acid residues at positions 216 and 225 are needed for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter.

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.

The studies have shown that the inner-coat oxalate decarboxylate (OxdD) has exposed an endogenous phytase, a commonly used feed for monogastric animals, in an active form at the spore surface. The potential of OxdD as a carrier protein is documented through the spore display of a bioactive heterologous passenger, the tetrameric [beta]-glucuronidase enzyme from Escherichia coli.

Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is anaturally competent bacterium that occupies various environments, including plant-associated, soil, and marineniches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43%of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. [ABSTRACT FROM AUTHOR]

Studies the influence of the DNA binding protein GerE on promoter activation in Bacillus subtilis. Sigma mutations in the Bacillus subtilis chromosome; Direct interaction of gerE with RNA polymerase subunits; Effect of alanine substitutions on transcription from the cotX and gerE promoters.

Examines the transcription from both o-A and O-H dependent promoters activated by Bacillus subtilis SpoOA. Identification of two amino acid substitutions in the carboxyl terminus of o-A, K356E and H359R; Effect of alanine substitutions in o-A and on o-A's ability to direct transcription in vivo and in vitro.