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.
Highlights
Bacterial spores are of high interest because of their extreme resistance properties, especially toward nutritional and environmental stresses (Rose et al, 2007), and the heterogeneity amongst them
The exponentially growing cells were subjected to growth in serial dilutions of 2x Schaeffer’s-glucose (2x SG) liquid medium (14N medium; further referred to as 14N cells) and a defined medium, buffered with MOPS supplemented with 15NH4Cl
These proteins like the ones mentioned above are present at higher abundance in the spores cultured on the solid agar plates. This suggests that the enhanced heat resistance is likely to be linked to the sum of specific cross-linked coat proteins and the degree of cross-linking. It appears that the spores developed on solid 2x SG agar plates and those in liquid defined medium in a shake flask, have a distinct coat protein composition, which is correlated, and likely functionally connected, to the difference in thermal resistance
Summary
Bacterial spores are of high interest because of their extreme resistance properties, especially toward nutritional and environmental stresses (Rose et al, 2007), and the heterogeneity amongst them. Due to their resistant nature, the methods to prevent bacterial contamination do not eliminate spores . The SASPs protect the DNA, while DPA forms a chelate complex with cations like calcium (Checinska et al, 2015) These chelate complexes are suggested to play a role in dehydration of the core, which in turn protects the spore from heat stress and UV light (Setlow et al, 2006). A peculiarity of these proteinaceous layers is that ∼30% of it is said to be composed of insoluble cross-linked proteins
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