Analysis of Bacillus subtilis spore germination and outgrowth in high-salinity environments

Abstract

Upon nutrient depletion, the soil bacterium Bacillus subtilis can form highly resistant, metabolically dormant spores. Spores consist of a dehydrated core (harboring the spore genome) enveloped in an inner spore membrane, a peptidoglycan germ cell wall and cortex, an outer spore membrane, and a proteinaceous coat. When specific nutrients (‘germinants’) become available again, they can bind to germinant receptors in the inner spore membrane and induce spore revival, consisting of a germination and an outgrowth phase. During germination, spores lose their resistance, release ions and Ca²⁺-dipicolinate (Ca²⁺-DPA) from the core in exchange for water (‘core rehydration’), and hydrolyze their cortex. When core rehydration is sufficient to allow enzymatic activity, metabolism is re-activated. This hallmarks the beginning of the outgrowth phase, during which spores undergo molecular reorganization and elongate. The effects of high salt concentrations and osmotic stress on spore revival were previously poorly investigated, although this topic is relevant for basic research, food microbiology, soil ecology, and astrobiology. Therefore, in this doctoral thesis, the impact of high salinity on Bacillus spore revival was examined, primarily focusing on B. subtilis spore germination in the presence of high NaCl concentrations. In general, increasing salt concentrations exerted increasingly detrimental effects on germination, although some spores initiated germination despite very high salinities. In the presence of high NaCl concentrations (≥ 1.2 mol/L), B. subtilis spore germination was delayed, slower, more heterogeneous, and less efficient. Other salts also inhibited germination, although their inhibitory strength varied depending on ion concentrations, ionic species (and their combination), and the chemical properties of the salt. Although ionic stress was indeed an important factor, high concentrations of non-ionic osmotic solutes had similar inhibitory strengths as iso-osmotic NaCl concentrations, suggesting that osmotic stress plays a decisive role in NaCl-inhibition. Strikingly, spores having strong coat defects showed exacerbated inhibition by NaCl but not by non-ionic solutes, indicating an important role of the spore coat (possibly in combination with the outer spore membrane) in protecting the subjacent inner spore structures (i.e. cortex, germ cell wall, germination apparatus, and inner spore membrane) from ionic stress. Based on these findings, a first mechanistic model for germination inhibition by high salinity is proposed. In this model, ionic interactions with the germinant and/or spore coat slow germinant passage to the germinant receptors, thereby delaying germination initiation. Subsequently, osmotic inhibition of core rehydration and concomitant Ca2+-DPA release slows germination after its initiation. While metabolic reactivation was observable at up to 4.8 mol/L NaCl, successful outgrowth in terms of elongation was observable at up to 2.4 mol/L NaCl, but only under nutrient-rich conditions. Transcriptomic analyses of salt-stressed outgrowing spores indicated many similarities to vegetative cells exposed to sustained high salinity, including the induction of B. subtilis’ complete genetic repertoire of osmoprotectant uptake and compatible solute synthesis. Taken together, this doctoral thesis yielded the first mechanistic model for inhibitory high-salinity effects on B. subtilis spore germination as well as the first comprehensive transcriptomics study of the salt stress response of outgrowing B. subtilis spores. These results contribute to the basic understanding of the influence of salt on B. subtilis’ life cycle, and are valuable for the aforementioned applied research fields as well.