Abstract
Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.
Highlights
Bacillus cereus, a member of the bacterial B. cereus sensu lato group, is a toxin-producing spore former which is widely distributed in the environment and commonly present in many raw foodstuffs, including rice, eggs, and milk
B. cereus spores are surrounded by a balloon-like exosporium, which is composed largely of spore-specific lipoproteins and glycoproteins; this exosporium layer is found in spores of all members of the B. cereus sensu lato group, including Bacillus anthracis [9,10]
Our results indicated that fusions of all GerR subunits and GerD are localized in the inner membrane (IM) of B. cereus spores based on the co-localization of protein fusions and the FM 4-64 membrane dye
Summary
A member of the bacterial B. cereus sensu lato group, is a toxin-producing spore former which is widely distributed in the environment and commonly present in many raw foodstuffs, including rice, eggs, and milk. These resistant spores can survive in improperly processed foods and germinate, grow, and produce toxins which can cause disease in humans [1,2,3,4]. B. cereus spores are surrounded by a balloon-like exosporium, which is composed largely of spore-specific lipoproteins and glycoproteins; this exosporium layer is found in spores of all members of the B. cereus sensu lato group, including Bacillus anthracis [9,10]. The various spore-specific structures and components all contribute to spore dormancy, as well as spores’ high resistance to agents, such as wet and dry heat, desiccation, UV and γ-radiation, and multiple types of toxic chemicals [11]
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