Visualization of Germination Proteins in Putative Bacillus cereus Germinosomes

Abstract

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.

Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions between GerD and Germinant Receptor Subunits in Bacillus cereus Spores

“…The GerR subunits A, B, and C and GerD were visualized in dormant B. cereus spores, using reporter proteins SGFP2 and mScarlet-I, as described in previous work [ 15 ]. The successful use of fluorescent reporter proteins to demonstrate germinosomes in B. cereus [ 15 ] suggests them to be functional.…”

“…The GerR subunits A, B, and C and GerD were visualized in dormant B. cereus spores, using reporter proteins SGFP2 and mScarlet-I, as described in previous work [ 15 ]. The successful use of fluorescent reporter proteins to demonstrate germinosomes in B. cereus [ 15 ] suggests them to be functional. A major aim of the new studies was to interrogate the interactions between each GerR GR subunit with the so-called germinosome scaffold protein GerD, using recombinant plasmids containing the C-terminus of the gene of interest fused to SGFP2 or mScarlet-I and controlled by the native promoter of the gene of interest ( Figure 1 and Table 1 ).…”

“…During spore formation, many proteins involved in spore germination, including a group of nutrient germinant receptors (GRs), are assembled and localized in the IM of the nascent spore or forespore [ 12 , 13 , 14 , 15 , 16 ]. The GRs allow dormant spores to continually monitor nutrients in their surroundings, as GRs respond to low-molecular-weight nutrients, commonly specific amino acids, sugars, and purine nucleosides to trigger germination.…”

“…Most importantly, the GR GerKB fused to mCherry and GerD fused to GFP co-localized in the IM foci in B. subtilis spores, and the clustering of GRs required all three GR subunits, as well as GerD [ 19 , 26 ]. However, interactions between GRs and GerD are poorly understood in B. subtilis spores, and there is no information about such interactions in B. cereus spores, although GR proteins and GerD also co-localize in a few foci in these spores [ 15 ].…”

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

“…The germinosome comprises germination receptors, scaffolding proteins and other components, such as the SpoVA protein involved in DPA transport ( Griffiths et al, 2011 ). Fluorescence visualization studies reveal that the germinosome localizes at one pole of the dormant endospore, then disperses as germination progresses ( Troiano et al, 2015 ; Breedijk et al, 2020 ; Wang et al, 2020 ). The germination cascade is then followed by degradation of the spore cell wall by lytic enzymes, increasing permeability and allowing Ca 2+ -DPA complexes and ions to escape the endospore in order to be replaced with water from the environment ( Griffiths et al, 2011 ; Wang et al, 2015 ).…”