Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Bacillus cereus can 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 of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores 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 of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores' IM. Previous studies have indicated that in spores of B. cereus ATCC 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 the gerR operon'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 ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~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 the gerD promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in Bacillus subtilis spores. Our data are the first evidence for the existence of germinosomes in B. cereus spores.