Mechanisms Underlying SleB Regulation During Spore Germination in Bacillus subtilis

Abstract

Bacterial endospores are formed by Bacillus and Clostridia in response to nutrient deprivation and can persist in the environment for a long period of time. Many endospore-forming species can cause severe diseases such as anthrax, food poisoning, tetanus, and gas-gangrene. However, endospores also play an important role in a variety of agricultural and industrial applications. Endospores can resist a variety of killing mechanisms including UV radiation, heat, desiccation, extreme pH, and antibiotics. Pathogenic spore formers pose a threat in nosocomial and clinical settings because spores are difficult to decontaminate. This work investigates the mechanism of activation of the Germination Specific Lytic Enzyme SleB and the role of YpeB in this process. It is highly important to understand the mechanisms of spore germination in more depth to develop novel targets that help in a more efficient spore decontamination. Genetic manipulation of Bacillus subtilis has led to the creation of a protease mutant strain lacking five proteases with a defective germination phenotype and a more stable YpeB during germination seen through germination assays and quantitative western blots. This stability correlated with a decrease in SleB activity seen in HPLC analysis of the muropeptides from dormant samples, and germinating samples after 30 and 60 minutes. The decrease in SleB-dependent muropeptide production during germination was quantified using mass spectrometry. Protein modeling and folding predictions accompanied with site-directed mutagenesis have led to the identification of the interacting regions between SleB and YpeB further characterizing the role of YpeB in SleB activation. Further work aiming at characterizing the role of SleB-YpeB complex formation on the spore inner membrane is required to better understand the mode of activation of SleB during germination.