Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes.

Bacillus cereuscan survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge ofB. cereusspores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features ofB. cereusspores are a core with the genetic material encased by multiple protective layers, an important one being the spores ' inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination ofB. cereusspores, we have examined the organization of germinant receptors (GRs) in spores ' IM. Previous studies have indicated that in spores ofB.cereusATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of thegerRoperon ' s promoter. Our results showed that: (i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; (ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and (iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in similar to 30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in similar to 85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of thegerDpromoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs inBacillus subtilisspores. Our data are the first evidence for the existence of germinosomes inB. cereusspores.