Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

Abstract

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.

FIG 1

Western blot analysis of protein extracts prepared from spores of B. cereus ATCC 14579 (WT) and congenic ΔcotE and ΔcotEΩcotE strains formed at 20°C and 37°C. Total spore proteins were extracted from the same amount of spores, fractionated by SDS-PAGE, and subjected to Western blot analysis using an anti-CotE polyclonal antibody (30). Black arrows indicate the CotE position, and gray arrows indicate CotE multimers. The asterisks indicate nonspecific bands.

FIG 2

Transmission electron microscopy of thin sections of spores of B. cereus ATCC 14579 WT (A and D), ΔcotE (B and E), and ΔcotEΩcotE (C and F) strains, formed at 20°C (A, B, and C) and 37°C (D, E, and F). c, spore coats; e, exosporium. The concentric layers of WT spores are intact and surround spores at both sporulation temperatures (A and D). ΔcotE spores formed at 20°C and 37°C exhibit a fragmented and detached exosporium (B and E), and the coat layers are locally interrupted (B). At both temperatures, some ΔcotEΩcotE spores exhibit the same concentric arrangement of the spore layers as WT, but some lack an intact exosporium and surrounding coats (C and F).

FIG 3

SDS-PAGE of the proteins extracted from the surface layers of B. cereus ATCC 14579 WT and ΔcotE spores. Coat and exosporium proteins were extracted from spores formed at 20°C and 37°C, separated on a 15% polyacrylamide gel, and stained with Coomassie brilliant blue. Arrows and numbers indicate bands in the protein extract of ΔcotE spores with enhanced (black arrows) or lowered (gray arrows) intensity compared to WT.

FIG 4

Hydrophobicity of spores of B. cereus ATCC 14579 WT (white bars), ΔcotE (black bars), and ΔcotEΩcotE (gray bars) strains formed at 20°C and 37°C. The percentage of hydrophobicity represents the proportion (×100) of spores in n-hexadecane after a separation into solvent and water phases. Each hydrophobicity percentage is the mean from three replicate experiments, each performed with an independently prepared spore suspension. Bars represent standard deviations. Different letters (a, b, c) show significant differences at P values of <0.05 (Tukey's test).

FIG 5

Germination in response to 0.5 mM inosine of B. cereus ATCC 14579 (■), ΔcotE (●), and ΔcotEΩcotE (▲) spores formed at 20°C (solid line) and 37°C (dashed line). The percentage of germination was determined after counting of phase-bright (ungerminated) and phase-dark (germinated) spores with a phase-contrast microscope. The numbers represent the means from three replicate experiments, each performed with an independently prepared spore suspension. Bars represent standard deviations.

FIG 6

Sensitivity of B. cereus ATCC 14579 (■), ΔcotE (●), and ΔcotEΩcotE (▲) spores formed at 20°C (solid line) and 37°C (dashed line) to wet heat at 90°C (A), UV-C (B), and pulsed light (C). Each log₁₀ reduction is the mean from three replicate experiments, each performed with an independently prepared spore suspension. Bars represent standard deviations.

FIG 7

Resistance of B. cereus WT (white bars), ΔcotE (black bars), and ΔcotEΩcotE (gray bars) spores formed at 20°C and 37°C exposed for 30 min to 5% H₂O₂ (vol/vol) (A), for 1 h to 7 U · ml⁻¹ lysozyme (B), and for 1 h to 250 µg · ml⁻¹ of mutanolysin (C). For each inactivation treatment, bars sharing the same letter (a, b, c, d) were not significantly different (P < 0.05, Tukey's test). Bars represent standard deviations for three experiments, each performed with an independently prepared spore suspension.