# Diverse Patescibacteria assemblages and prevalence of ultra-small free-living Parcubacteria along a subterranean estuary

## TL;DR

This study explores the distribution and diversity of ultra-small Patescibacteria in a coastal groundwater system, revealing their adaptation to different salinity levels and highlighting their ecological significance.

## Contribution

The study provides new insights into the niche partitioning and high novelty of Patescibacteria along salinity gradients in subterranean estuaries.

## Key findings

- Patescibacteria taxa adapt to varying salinity conditions in groundwater, occupying fresh, brackish, and saline niches.
- Ultra-small Parcubacteria cells increase in abundance from fresh to saline groundwater and are visually identified.
- Most detected taxa show high novelty, with less than 95% similarity to known sequences.

## Abstract

Patescibacteria are a group of novel, mostly uncultivated bacteria characterized by ultra-small cell sizes and streamlined genomes. They are ubiquitous in diverse ecosystems, often prevailing in subsurface environments, yet basic aspects such as variability in cell size, abundance, and niche preferences of different taxa within Patescibacteria remain unknown, particularly along salinity gradients. Combining flow cytometry, catalyzed reporter deposition-fluorescence in situ
hybridization (CARD-FISH), and 16S rRNA gene sequencing, we characterized Patescibacteria assemblages along a Mediterranean subterranean estuary resulting from the mixing of fresh groundwater with seawater. Patescibacteria occupied the entire subterranean salinity gradient through the replacement of taxa prevailing in fresh (Magasanikbacteria, Jorgensenbacteria UBA9983), brackish (Portnoybacteria, Yanosfkybacteria, and Peribacteria), and saline groundwater (Nomurabacteria, unidentified Gracilibacteria). Most of the detected ASVs showed less than 95% similarity to their closest match, pointing to high novelty within coastal groundwater Patescibacteria. Flow cytometry unveiled a clear population of ultra-small prokaryotes that increased in abundance from fresh to saline groundwater, and which coincided with the presence of free-living minute coccoid cells identified as Parcubacteria by CARD-FISH. Some symbiotic-like associations with prokaryotes and eukaryotes were also observed, at least within Parcubacteria. These results provide one of the rare visual observations of Patescibacteria, and the substantial diversity of yet-unidentified taxa suggests an overlooked importance of this group in coastal groundwater.

Patescibacteria are an enigmatic group of bacteria of ultra-small sizes and reduced genomes, commonly found in subsurface environments but largely unexplored in terms of their ecological roles. Despite being present in both freshwater and marine systems, no study has explored how they distribute along salinity gradients. This study provides new insights into their distribution, diversity, and niche partitioning along a Mediterranean subterranean estuary characterized by a strong salinity gradient. We show that Patescibacteria taxa seem to adapt to varying groundwater salinity conditions, displaying a remarkable capacity to occupy fresh, brackish, and saline niches through changes in composition. The identification of ultra-small coccoid cells and symbiotic-like associations highlights a diversity of lifestyles within these groups and provides one of the scarce visual proofs of Patescibacteria. With most detected taxa being highly novel, these findings point to an overlooked importance of Patescibacteria in coastal aquifers, biogeochemically active sites ubiquitous along most coastlines.