The genome sequence of the Gram-positive soil bacterium Bacillus subtilis was completed in 1997 (Kunst et al., 1998) and the results included the identification of a putative transcription unit encompassing the yloI to yloS genes. Within this region of the B. subtilis chromosome 11 putative open reading frames were found with a wide diversity of probable functions. In this work we have analyzed transcription in the region of the priA-cpgA genes and we have mapped a promoter which is located inside the priA gene and its activity directs transcription of the def-yloM genes. Moreover, this transcript can be extended at low level to the prpC-priK-cpgA genes. Analysis of the sequence in proximity of the transcription start site revealed a sequence suitable for the housekeeping σA subunit of RNA polymerase. Analysis of the β-glactosidase activity of transcription fusions revealed that the identified promoter is active at low level and its activity is increased during late exponential phase of growth.
The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently, there are no technical possibilities to predict whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study, we compared four variants of B. subtilis spores presenting a fragment of a FliD protein from Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their own phagocytosis and activate both, the J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that the observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent of its abundance and localization in the spore coat structure.
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