Loss of PIKfyve Causes Transdifferentiation of Dictyostelium Spores Into Basal Disc Cells

The 1-phosphatidylinositol-3-phosphate 5-kinase PIKfyve generates PtdIns3,5P2 on late phagolysosomes, which by recruiting the scission protein Atg18, results in their fragmentation in the normal course of endosome processing. Loss of PIKfyve function causes cellular hypervacuolization in eukaryotes and organ failure in humans. We identified pikfyve as the defective gene in a Dictyostelium mutant that failed to form spores. The amoebas normally differentiated into prespore cells and initiated spore coat protein synthesis in Golgi-derived prespore vesicles. However, instead of exocytosing, the prespore vesicles fused into the single vacuole that typifies the stalk and basal disc cells that support the spores. This process was accompanied by stalk wall biosynthesis, loss of spore gene expression and overexpression of ecmB, a basal disc and stalk-specific gene, but not of the stalk-specific genes DDB_G0278745 and DDB_G0277757. Transdifferentiation of prespore into stalk-like cells was previously observed in mutants that lack early autophagy genes, like atg5, atg7, and atg9. However, while autophagy mutants specifically lacked cAMP induction of prespore gene expression, pikfyve− showed normal early autophagy and prespore induction, but increased in vitro induction of ecmB. Combined, the data suggest that the Dictyostelium endosomal system influences cell fate by acting on cell type specific gene expression.

The choice of anchoring protein influences interaction of recombinant Bacillus spores with the immune system

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 FliD protein of Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their phagocytosis and activate both, J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent on its abundance and localization in the spore coat structure.

The C-Terminal Zwitterionic Sequence of CotB1 Is Essential for Biosilicification of the Bacillus cereus Spore Coat

ABSTRACTSilica is deposited in and around the spore coat layer ofBacillus cereus, and enhances the spore's acid resistance. Several peptides and proteins, including diatom silaffin and silacidin peptides, are involved in eukaryotic silica biomineralization (biosilicification). Homologous sequence search revealed a silacidin-like sequence in the C-terminal region of CotB1, a spore coat protein ofB. cereus. The negatively charged silacidin-like sequence is followed by a positively charged arginine-rich sequence of 14 amino acids, which is remarkably similar to the silaffins. These sequences impart a zwitterionic character to the C terminus of CotB1. Interestingly, thecotB1gene appears to form a bicistronic operon with its paralog,cotB2, the product of which, however, lacks the C-terminal zwitterionic sequence. A ΔcotB1B2mutant strain grew as fast and formed spores at the same rate as wild-type bacteria but did not show biosilicification. Complementation analysis showed that CotB1, but neither CotB2 nor C-terminally truncated mutants of CotB1, could restore the biosilicification activity in the ΔcotB1B2mutant, suggesting that the C-terminal zwitterionic sequence of CotB1 is essential for the process. We found that the kinetics of CotB1 expression, as well as its localization, correlated well with the time course of biosilicification and the location of the deposited silica. To our knowledge, this is the first report of a protein directly involved in prokaryotic biosilicification.IMPORTANCEBiosilicification is the process by which organisms incorporate soluble silicate in the form of insoluble silica. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification was not studied until recently. We previously demonstrated that biosilicification occurs inBacillus cereusand its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acid. The present study reveals that aB. cereusspore coat protein, CotB1, which carried a C-terminal zwitterionic sequence, is essential for biosilicification. Our results provide the first insight into mechanisms required for biosilicification in prokaryotes.

Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores

Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from<i> Clostridium thermocellum</i> was expressed, identified and displayed on the surface of <i>Bacillus subtilis </i>spores by using the spore coat protein G of <i>B. subtilis </i>as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40°C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.