# Early activation of bioenergetic metabolism powers bacterial spore germination

## TL;DR

Bacterial spores start using energy metabolism early during germination, challenging the idea that germination is purely passive.

## Contribution

The study reveals that bioenergetic processes begin at the onset of germination, not after hydration.

## Key findings

- Energization of cytoplasmic metabolism and the electron transport chain occurs early in germination.
- The Yth isoform of the bd oxidase enables rapid electron transfer when other oxidases are hindered.
- Deleting Yth slows germination, linking bioenergetics to the germination process.

## Abstract

Bacterial spores can survive, dormant, for thousands of years and yet are able to germinate into vegetative cells in about an hour. The current consensus is that resumption of metabolism is a late event in germination occurring only once the spores have sensed their germinants and rehydrated. Here, a biophysical approach called remission spectroscopy is used that measures haem groups within energy-transducing cytochrome enzymes of intact, germinating spores. We find that energy metabolism actually starts right at the beginning of the germination process. Our observations upturn the view that germination is only a passive process of solutes flowing down concentration gradients. Instead, we see active energization of the metabolic system that can be powering processes such as transport or macromolecular synthesis.

Dormant bacterial spores germinate to become vegetative cells upon germinant exposure. Despite many germinants being energy sources, bioenergetic processes have been overlooked as germination can proceed, albeit slowly, without exogenous energy sources. Here, we apply remission spectroscopy to noninvasively measure energization of the electron transport chain (ETC) in germinating spores. In Bacillus megaterium and Bacillus subtilis, energization of cytoplasmic metabolism and the ETC occurs early in germination, before or alongside water ingress and bulk CaDPA efflux. The aa3-type oxidases (Qox, Cta) accumulate nonradical ferryl intermediates of their catalytic cycle, slowed by a high membrane potential. The Yth isoform of the bd oxidase, present in spores, allows rapid electron transfer to O2 when the aa3-type oxidases are hindered, establishing a role for this enzyme. Deletion of Yth slows the initiation of “absorbance”/attenuance loss, directly linking bioenergetic processes to germination. We propose a powered germination model, where the Ger-mediated signaling cascade and bioenergetic processes occur in parallel and are mutually influenced by each other. This model explains why germination on energy-rich molecules (e.g., glucose) is often much faster than on energy-poor ones (e.g., KBr).