Exploring the bioenergetics of bacterial spore germination using Bacillus megaterium

When confronted with unfavourable growth conditions, Bacillus and Clostridium species initiate the process of sporulation whereby a bacterial cell undergoes drastic reconfiguration to become a robust, multi-layered, dormant spore. When the availability of nutrients is sensed via germinant receptors, Bacillus spores can exit dormancy (germinate) and return to metabolically active life. Spore germination unfolds on a much faster timescale than sporulation, and many aspects of this process are not fully understood – essential events during germination are localised in the spore inner membrane but this is not easily accessible for biochemical experimentation because of the spore’s multi-layered architecture. In this work, Bacillus megaterium QM B1551 was used as a model to optimise a protocol for producing high quality membrane preparations from medium-scale spore cultures. A chromatographic method was developed to enable extraction of large membrane protein complexes (e.g. germinant receptors) directly from their atypical spore environment for biochemical/structural characterisation. NADH:O2 reaction assays, native gel electrophoresis and proteomic analyses on isolated spore membranes revealed the composition of an active, branched membrane-bound electron transport chain present in dormant spores. Two uncharacterised, spore-specific paralogues were found: a type II NADH dehydrogenase (YumB not Ndh) and cyt bd oxidase (YthAB not CydAB). To understand when germinating spores resumed glucose-powered oxidative metabolism using this ETC, traditional spore germination assays, enzymatic detection of glucose consumption and H2O2 production, O2 consumption measurements and novel haem remission spectroscopy experiments were carried out. Based on these, a revised timeline of events in germination was proposed, where glucose serves as both a signalling molecule and an exogenous energy source, oxidative metabolism and germination are initiated concurrently, and electron transport precedes detectable germination – YthAB is critical for resumption of bioenergetics in germinating spores. Lastly, a serendipitous cryo-EM map of the enzyme succinate dehydrogenase was refined from crudely enriched membranes of B. subtilis cells.