Investigation of Bacillus subtilis spore germination at high salinity

Abstract

The effects of high salt concentrations on the germination of Bacillus subtilis endospores are barely investigated. This thesis addressed questions concerning high-salinity effects on spore germination, the roles of cellular components in germination under high salt conditions and salt-dependent alterations in gene expression during spore outgrowth. B. subtilis wild type spores are generally able to germinate despite the presence of very high salt concentrations and osmolalities (up to 3.6M and 7.2 osmol/kg, respectively). However, elevated salt concentrations exert inhibitory effects on germination, leading to a delay of germination onset and decreased germination efficiency. Four major factors were identified to differentially affect spore germination at high salinity: the germination medium, the employed germination trigger, the type of ionic species and the ion concentration. Cationic and anionic species, valence and general chemical properties seem to influence the inhibitory features of a salt, whereas the overall osmolality of the germination medium is apparently less important. Salt inhibition seems to have several targets, at least one being related to nutrient germination receptor (GR) functionality. In fact, the different GRs were found to be unequally affected by different salts. However, high salinity can also inhibit non-nutrient germination that does not involve GRs, indicating at least one additional, GR-independent inhibition target. The germination analyses of mutant spores provided interesting information on the involvement of different cellular components within the germination process. The anionic phospholipid cardiolipin (CL) is not essential, but beneficial for successful nutrient germination in the absence of salt. Yet, CL is highly important for germination in a high-salinity environment, possibly due to stabilizing effects on the GRs. The K⁺-transport systems KtrAB and KtrCD are not essential for germination, but KtrAB seems to play a role for germination in the presence of high NaCl concentrations. Regardless of the salinity of the germination medium, the five osmoprotectant uptake systems (OpuA to OpuE) are not involved in germination, although they generally seem to facilitate outgrowth and subsequent growth.