Just add water? What happens to bacterial RNA during desiccation?

What started as a simple question, ‘How do non-spore forming bacteria cope with desiccation?’ has evolved into a newly funded NSF award.

The desert soils in Southern Arizona are scorching hot in the summer. For seven consecutive days in July 2020 a soil temperature and moisture probe at one of our field sites recorded a 5 cm soil temperature of 50ºC (122ºF) with an extremely low soil moisture (~2% volumetric water content).

These hot and dry conditions mummify surface soil microbes in place where they persist in desiccation-induced dormancy awaiting the monsoon rains. A large portion of these microbes are non-spore formers. Although the gene regulation cascades leading to dormancy are well-understood in spore forming bacteria, the transitions to and from desiccation-induced dormancy in diverse non-spore forming bacteria are less well described.

We isolated a bunch (~3,000) of microbes from local soils and started to investigate how survivability, internal metabolites (in collaboration with JGI), and gene expression are influenced by desiccation. What follows is a sneak peek of the data and a brief outline of where we’re going with it and what the implications are.

Our preliminary data that show that even dry soil bacteria contain substantial amounts of measurable RNA. But during the drying process, some of the intracellular RNA is degraded. We have two observations that support this: 1) all 4 ribonucleosides (ACGU) increase dramatically relative to controls; and 2) measurable RNA levels decrease to about 30-50% less RNA in desiccated cells, relative to hydrated controls.

This putative RNA degradation occurs concomitantly with a loss in survival of dried cells relative to hydrated cells. Occam's razor would suggest that this reduction in RNA is likely the result of cell death. And that’s what we initially thought.

But dead is DEAD. Can you imagine a mummified human coming back to life if you just add water? I know who I’d call!

The dried cells are not dead. Something interesting happens when we rehydrate the cells with water vapor instead of liquid water (as is the case in the viability assays). Cell survivability comes back to 100%. That is, the survival of desiccated cells that are gently rehydrated with water vapor is the same as cells that were never dehydrated.

Interestingly, ribonucleosides and RNA content return to levels indistinguishable from cells that were incubated alongside the desiccated samples but had never been desiccated.

We had an idea that perhaps some intracellular RNAs may be more stable than others. So we wrote a proposal about it.

And it was funded.

We found out in November that for the next 5 years we'll be working with NSF with our CAREER award to investigate how and if certain RNAs are stabilized in desiccated dormant soil bacteria and how RNA stability plays a role in desiccation tolerance. As part of the award, we’ll also develop a graduate course that teaches students the pillars of effective scientific mentorship. Grad students working on the project will take this class and have genuine mentorship opportunities in our lab to guide undergrads in collecting some of the data.

RNA is often touted as the ‘gold standard’ molecule for microbial activity. This is true for growing cells, where RNAs are turned over rapidly. But we know very little about what happens to RNA in non growth states other than spores. So our research could influence how metatranscriptomes or RNA:DNA ratios are interpreted. Do inactive dry cells harbor RNA? Are these transcripts physiologically meaningful? There’s lots to learn and we’re excited to get started!

The project was started by Dr. Ryan Bartelme with assistance from undergrad Brad Schlottman shortly before they moved on to greener pastures in 2019. The key data was obtained by our rockstar MS student Izzy Viney throughout the pandemic.

In what's been a tough, tough, tough year (or 2) for me, our lab group, and everyone else, we’re feeling lucky, privileged, and grateful that the stars aligned for this award. We'll be looking for folks to join us in various capacities in the coming months. Be sure to subscribe to Un-Cultured to stay on top of these openings!

There's also some more good news to come that I can't share quite yet. 😉