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.