The physiological function for thiaminase II, a thiamin-degrading enzyme, has eluded investigators for more than 50 years. Here, we demonstrate that this enzyme is involved in the regeneration of the thiamin pyrimidine rather than in thiamin degradation, and we identify a new pathway involved in the salvage of base-degraded forms of thiamin. This pathway is widely distributed among bacteria, archaea and eukaryotes. In this pathway, thiamin hydrolysis products such as N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (formylaminopyrimidine; 15) are transported into the cell using the ThiXYZ transport system, deformylated by the ylmB-encoded amidohydrolase and hydrolyzed to 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP; 6)-an intermediate on the de novo thiamin biosynthetic pathway. To our knowledge this is the first example of a thiamin salvage pathway involving thiamin analogs generated by degradation of one of the heterocyclic rings of the cofactor.

The ban on feed antibiotics by more and more countries, and the expected ban on ZnO in feed supplementation from 2022 in the EU, urge researchers and pig producers to search for new alternatives. One possible alternative is to use the so-called “next-generation probiotics (NGPs)” derived from gastrointestinal tract.

The cyanobacterium Calothrix sp. PCC 7601 can adapt its pigment content in response to changes in the incident light wavelength. It synthesizes, as major light‐harvesting pigments, either phycocyanin 2 (PC2, encoded by the cpc2 operon) under red light or phycoerythrin (PE, encoded by the cpeBA operon) under green light conditions. The last step of the signal transduction pathway is characterized by a transcriptional control of the expression of these operons. Partially purified protein extracts were used in gel retardation assays and DNase I footprinting experiments to identify the factors that interact with the promoter region of the cpeBA operon. We found that two proteins, RcaA and RcaB, only detected in extracts of cells grown under green light, behave as positive transcriptional factors for the expression of the cpeBA operon. Treatment of the fractions containing RcaA and RcaB with alkaline phosphatase prevents the binding of RcaA but not of RcaB to the cpeBA promoter region. A post‐translational modification of RcaA thus modulates its affinity for DNA.

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 a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, 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.

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.

A's ability to direct transcription in vivo and in vitro. We found that alanine substitutions at these positions specifically reduced expression from the A -dependent, Spo0A-dependent promoters, spoIIG and spoIIE, in vivo. Furthermore, we found that stimulation of spoIIG promoter activity by Spo0A in vitro was reduced by the single substitutions H359A and H359R in A .A growing body of evidence supports the idea that sitespecific DNA binding proteins activate promoters in bacteria by contacting RNA polymerase (reviewed in reference 4). The site on RNA polymerase that is contacted by the activator protein appears to depend in part on the site on DNA at which the activator protein is bound (16). The Escherichia coli cyclic AMP receptor protein (CAP) interacts with the C-terminal domain (CTD) of the RNA polymerase alpha subunit when CAP is bound at a site centered about 61 bp upstream from the transcription start point (Ϫ61) (reviewed in reference 11), whereas CAP bound at a site centered at about position Ϫ41 activates transcription by contacting the N-terminal domain of the ␣ subunit (25; reviewed in reference 5). Other activator proteins contact the subunit of RNA polymerase. FNR (a protein that is structurally similar to CAP), PhoB, MalT, and cI from phage lambda bind to sites that overlap the Ϫ35 region of promoters, and probably contact the subunit of RNA polymerase (reference 5 and references therein; 9, 22-24). Some promoters contain multiple binding sites for activators. In some of these cases the two coactivators appear to interact directly with the RNA polymerase (6,18,29). At other promoters one coactivator affects DNA binding by the second coactivator, which is the only one to make direct contact with RNA polymerase (26).Spo0A from Bacillus subtilis, which is required for the initiation of sporulation, binds the spoIIG promoter at two sites known as 0A boxes, centered at positions Ϫ37 and Ϫ87 (reviewed in reference 15; 1, 27). Binding of Spo0A at these sites stimulates utilization of the spoIIG promoter by RNA polymerase containing A , a homolog of E. coli 70 (1, 3, 20, 27, 28). It is not known whether spoIIG promoter activation involves interactions between Spo0A and RNA polymerase. Baldus et al. (2) found that spoIIG promoter activity was reduced in mutants of B. subtilis in which A contained one of two single amino acid substitutions (glutamate substituted for lysine at position 356, K356E, or arginine substituted for histidine at position 359, H359R). These observations led to the suggestion that spoIIG promoter activation involves an interaction between Spo0A and A . The two amino acid substitutions in A that reduced spoIIG promoter activity lie near the region of A that makes sequence-specific contacts with the base pairs at the Ϫ35 region of its cognate promoters (Fig. 1). Other amino acid substitutions in this region of A and other sigma factors have been shown to change the specificity of promoter utilization by RNA polymerase containing these mutant sigma factors (12,19,32). An alternative e...

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).

The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric ␤-glucuronidase enzyme from Escherichia coli.Under extreme nutrient deprivation, Bacillus subtilis has the ability to enter a complex differentiation process that culminates with the formation of an extremely resistant spore.