The Influence of Sporulation Conditions on the Spore Coat Protein Composition of Bacillus subtilis Spores

Abstract

Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions, especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared under a liquid medium condition. It has also been shown that the more mature a spore is, the higher is its heat resistance most likely mediated, at least in part, by the progressive cross-linking of coat proteins. The current study for the first time assesses, at the proteomic level, the effect of two commonly used sporulation conditions on spore protein presence. ¹⁴N spores prepared on solid Schaeffer's-glucose (SG) agar plates and ¹⁵N metabolically labeled spores prepared in shake flasks containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The former condition mimics the industrial settings while the latter conditions mimic the routine laboratory environment wherein spores are developed. As seen previously in many studies, the spores prepared on the solid agar plates show a higher thermal resistance than the spores prepared under liquid culture conditions. The ¹⁴N:¹⁵N isotopic ratio of the 1:1 mixture of the spore suspensions exposes that most of the identified inner coat and crust proteins are significantly more abundant while most of the outer coat proteins are significantly less abundant for the spores prepared on solid SG agar plates relative to the spores prepared in the liquid MOPS buffered defined medium. Sporulation condition-specific differences and variation in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that the coat protein cross-linking may also be condition specific. Since the core dipicolinic acid content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Corroborating the proteomic analyses, electron microscopy analyses show a significantly thinner outer coat layer of the spores cultured on the solid agar medium.

Keywords: Bacillus; proteomics; quantitative proteomics; spores; sporulation conditions.

FIGURE 1

Workflow of quantitative proteomics of spores prepared under the most common sporulation conditions used in industries (solid agar plates) and in routine microbiology laboratories (liquid medium). The magenta color indicates presence of ¹⁵N in to the medium, cells, and therefore also in spores.

FIGURE 2

Fold changes in spore coat protein expression in spores cultured on solid agar plates and in shake flask with liquid medium. The bars indicate the mean ratios of proteins across four replicates while error bars indicate the standard deviations in these ratios. By definition, the values are within 95.40% confidence interval in the range of the error bars. The protein localization is based upon their sigma factor regulations and the available literature (van Ooij et al., 2004; Imamura et al., 2010; McKenney et al., 2010; Driks and Eichenberger, 2016). Cs, Crust; OC, Outer coat; IC, Inner coat; Ct. asso., Spore coat associated proteins; OM, Outer membrane; Cx, Cortex; IM, Inner membrane; Cr, Core; Sp. asso., Spore associated proteins.

FIGURE 3

Thermal resistance of spores developed under different sporulation conditions. Log₁₀ values of colony forming units (CFU) obtained after 48 h incubation per OD₆₀₀ of spores. The values represent the mean averages for three biological replicates where the error bars represent the standard deviation observed for two technical replicates. By definition, the values are within 95.40% confidence interval in the range of the error bars.

FIGURE 4

Core dipicolinic acid (DPA) content as % per spore dry weight of spores developed under different sporulation conditions. The error bars indicate the standard deviation within two technical replicates. By definition, the values are within 95.40% confidence interval in the range of the error bars.

FIGURE 5

Phase contrast microscopic images of spores cultured on solid agar plates (A) and in shake flasks containing liquid medium (B). The spores are observed under 100× magnification with oil under simple phase contrast microscope. Scale bars indicate 10 μm.

FIGURE 6

Appearance of liquid suspensions of spores cultured on 2x SG agar plates (A) and in 3-(N-morpholino) propane sulfonic acid (MOPS) buffered defined liquid medium in shake flask (B). Two spore crops differ in their color as liquid suspensions in sterile Milli-Q water with equal OD₆₀₀.

FIGURE 7

Electron microscopic images of spores developed under solid (A) and liquid (B) medium conditions with the corresponding thickness of the spore coat layers (C). The coat layer thicknesses are averaged over 80 individual spores. The error bars indicate the standard deviations between two biological replicates. By definition, the values are within 95.40% confidence interval in the range of the error bars. Cr, core; Cx, cortex; Ic, inner coat; Oc, outer coat.