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.

Investigating the influence of spore maturation and sporulation conditions on MalS expression during Bacillus subtilis spore germination

“…Spores cultured in MOPS liquid medium and on 2x SG solid medium were harvested after 48 and 96 hrs 14 . Spore samples were extensively washed with pre-chilled milliQ-water at 4°C.…”

“…The harvested spores were examined under the microscope using a haemocytometer and the spore yield was estimated. Remaining vegetative cells and/or germinated cells (phase dark) were removed using the Histodenz gradient centrifugation method describe by Abhyankar et al 14 . The purified harvested spores contained more than 99% phase bright spores.…”

“…The above-mentioned studies however, differ with respect to the age of the spores used in each study and the medium used for sporulation. Previously, it has been reported that the sporulation conditions and maturation time affect the spore characteristics and resistance properties 14 . Spores formed on solid rich media are generally more heat resistant and are characterised by a higher degree of coat proteins involved in cross-linking 14 .…”

“…Previously, it has been reported that the sporulation conditions and maturation time affect the spore characteristics and resistance properties 14 . Spores formed on solid rich media are generally more heat resistant and are characterised by a higher degree of coat proteins involved in cross-linking 14 . It has also been shown that higher spore coat protein cross-linking correlates with slight delayed germination times 11 .…”

Electrical-charge accumulation enables integrative quality control duringB. subtilissporulation

“…For instance, endospores produced at high temperature (~50 °C) exhibit higher resistance against heat (~100 °C) (20). The thickness of spore coats varies depending on the culture medium (21). Strikingly, even with an identical medium and temperature, the endospores prepared in liquid are less resistant to heat than the endospores produced on plates (22).…”