The Study of Diversity in Sporulation among Closely Genetically Related <i>Bacillus cereus</i> Strains

Abstract

Bacillus cereus is an important foodborne pathogenic bacterium. Although several B. cereus strains have been isolated from the environment, the differences among these strains with respect to spore formation ability and cell morphology need clarification. In this study, a phylogenetic tree was constructed based on the 16S rRNA gene sequences of nine strains of B. cereus. Spore formation and morphology of these nine strains were compared using both phase-contrast and fluorescence microscopy to create an index of the designated sporulation stages. Additionally, to investigate the efficiency of heat-resistant spore formation. Phylogenetic analysis revealed that five strains (ATCC 14579 T , NBRC 3457, NBRC 3514, NBRC 3836, and NBRC 13597) clustered together and the remaining four (ATCC 10987, NBRC 3003, NBRC 13494, and NBRC 13690) were genetically distinct from each other. Phase-contrast microscopy revealed significant differences in the sporulation stages among the nine strains. Furthermore, the efficiency of heat-resistant spore formation also differed, even among genetically related strains. In conclusion, a variety of cell morphologies during sporulation were observed among the nine B. cereus strains. We propose a designation of sporulation stages in B. cereus ATCC 14579 T , which may be used as an index for evaluating the sporulation progress of B. cereus.

SpoIVA is an essential morphogenetic protein for the formation of heat- and lysozyme-resistant spores in Clostridium sporogenes NBRC 14293

“…In the present study, we defined the sporulation stage based on the morphological changes in cells as observed using phase-contrast and fluorescence microscopy in C. sporogenes NBRC 14293. These morphological changes are similar to those described for B. subtilis , B. cereus , and C. botulinum ( Errington, 1993 ; Al-Hinai et al, 2015 ; Hosomi et al, 2015 ; Kuwana et al, 2022 ). Using TEM, we also analyzed the structure of C. sporogenes NBRC 14293 mature spores, revealing the presence of a core, cortex, spore coat, and exosporium.…”

“…Cell membranes and DNA were stained with NAO and Hoechst 33342, respectively. We observed and compared the different sporulation stages of C. sporogenes NBRC 14293 with those identified in other endospore-forming organisms, such as B. subtilis 168, B. cereus ATCC 14579, and C. botulinum type B strain 111 ( Nicholson and Setlow, 1990 ; Errington, 1993 ; Hosomi et al, 2015 ; Kuwana et al, 2022 ). Rod-shaped cells, with DNA enclosed in a cytoplasmic membrane without asymmetric division, indicated the progression from vegetative growth to stage II sporulation ( Figure 4A ).…”

“…In B. subtilis , we have previously demonstrated that auramine O effectively stained the FS from stage VI and mature spores, whereas APBT stained the membranes of vegetative cells and MCs and the periphery of the FS and mature spores ( Kuwana et al, 2022 ). In the present study, we stained the wild-type and spoIVA : erm mutant strains of C. sporogenes during sporulation and observed APBT fluorescence in the cellular membrane and FS of the wild-type and spoIVA : erm mutant cells ( Figures 5B, G ).…”

“…After 24 or 48 h of growth in GAM agar at 37°C, C. sporogenes cells were stained using acridine orange 10-nonyl bromide (nonyl acridine orange, NAO) (final concentration 0.01 mg/ml) (Santa Cruz Biotechnology, Dallas, TX, USA), Hoechst 33342 (final concentration 0.01 mg/ml) (Thermo Fisher Scientific, Waltham, MA, USA), 2-(4-aminophenyl)benzothiazole (APBT) (final concentration 0.01 mg/ml) (Tokyo Chemical Industry, Tokyo, Japan), auramine O (final concentration 0.01 mg/ml) (Tokyo Chemical Industry), or pararosaniline hydrochloride (final concentration 0.01 mg/ml) (Nacalai Tesque, Inc., Kyoto, Japan). The cells were incubated with the products in 10 mM Tris–HCl (pH 7.6) for 10 min at 25°C, and the samples were transferred onto microscope slides as previously described ( Kuwana et al, 2022 , 2023 ). Pararosaniline hydrochloride is a fluorescent probe used for the spectrofluorometric determination of ondansetron, an antiemetic drug ( Essawy and Ali, 2018 ).…”