# Thermophilic bacteria employ a contractile injection system in hot spring microbial mats

## TL;DR

Thermophilic bacteria in hot springs use contractile injection systems, which were studied using advanced imaging and sequencing techniques.

## Contribution

A novel approach combining cryoET and bioinformatics reveals CIS production in natural thermophilic bacteria.

## Key findings

- Thermophilic Chloroflexota bacteria produce intracellular CIS particles in hot spring mats.
- CIS production is niche-specific in structured microbial communities.
- CIS lineage in Chloroflexota/Deinococcota shares similarities with Streptomyces cytoplasmic CIS.

## Abstract

Bacterial contractile injection systems (CISs) are multiprotein complexes that facilitate the bacterial response to environmental factors or interactions with other organisms. Multiple novel CISs have been characterised in laboratory bacterial cultures recently; however, studying CISs in the context of the native microbial community remains challenging. Here, we present an approach to characterise a bioinformatically predicted CIS by directly analysing bacterial cells from their natural environment. Using cryo-focused ion beam milling and cryo-electron tomography (cryoET) imaging, guided by 16S rRNA gene amplicon sequencing, we discovered that thermophilic Chloroflexota bacteria produce intracellular CIS particles in a natural hot spring microbial mat. We then found a niche-specific production of CIS in the structured microbial community using an approach combining metagenomics, proteomics, and immunogold staining. Bioinformatic analysis and imaging revealed CISs in other extremophilic Chloroflexota and Deinococcota. This Chloroflexota/Deinococcota CIS lineage shows phylogenetic and structural similarity to previously described cytoplasmic CIS from Streptomyces and probably shares the same cytoplasmic mode of action. Our integrated environmental cryoET approach is suitable for discovering and characterising novel macromolecular complexes in environmental samples.

Graphical Abstract