Abstract
To facilitate more accurate spore proteomic analysis, the current study focuses on inducing homogeneous sporulation by overexpressing kinA and assesses the effect of synchronized sporulation initiation on spore resistance, structures, the germination behavior at single-spore level and the proteome. The results indicate that, in our set up, the sporulation by overexpressing kinA can generate a spore yield of 70% within 8 h. The procedure increases spore wet heat resistance and thickness of the spore coat and cortex layers, whilst delaying the time to spore phase-darkening and burst after addition of germinant. The proteome analysis reveals that the upregulated proteins in the kinA induced spores, compared to spores without kinA induction, as well as the ‘wildtype’ spores, are mostly involved in spore formation. The downregulated proteins mostly belong to the categories of coping with stress, carbon and nitrogen metabolism, as well as the regulation of sporulation. Thus, while kinA overexpression enhances synchronicity in sporulation initiation, it also has profound effects on the central equilibrium of spore formation and spore germination, through modulation of the spore molecular composition and stress resistance physiology.
Highlights
Spore forming bacteria, such as Bacillus subtilis, develop into an endospore when environmental conditions are unfavorable
In the current set of experiments, B. subtilis gene kinA has been artificially induced to synchronize the expression of KinA in the phosphorelay regulating spore formation, while the glucose concentration in the environment is kept at 40mM, to halt the initiation of sporulation due to catabolite repression on Spo0A phosphorylation induced by high glucose concentration [21]
This artificial sporulation induction (ASI) resulting in the synchronization of sporulation initiation results in the formation of a high percentage of spores in a short time (70% spores within 8 h), but does not alter the final yield of 80% spores, which is comparable to the wildtype strain used as a reference
Summary
Spore forming bacteria, such as Bacillus subtilis, develop into an endospore when environmental conditions are unfavorable. The initiation of sporulation takes place heterogeneously It is controlled by a phosphorelay system, which is in charge of phosphorylating Spo0A. To facilitate a more sensitive spore proteomic analysis, a kinA-inducible strain of Bacillus subtilis may be used to synchronize input of phosphoryl groups and trigger sporulation [12,14,17,18]. The current study focuses on synchronizing the initiation of sporulation by overexpressing kinA, synchronizing the expression of KinA, and controlling the medium glucose concentration, where high glucose represses, and low glucose derepresses, the phosphorylation of Spo0A. While kinA overexpression through ASI enhances the synchronicity in sporulation initiation, it has profound effects on the central equilibrium of spore formation and spore germination through the modulation of the spore molecular composition and stress resistance physiology
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