Kueselia aquadivae gen. nov., sp. nov., the first member of the family Isosphaeraceae isolated from subsurface percolates

Abstract

Subsurface habitats, found under various geological conditions, exhibit diverse microbial communities. The vadose zone, a previously unexplored subsurface compartment, connects the surface to phreatic groundwater. Drilling into the subsurface allows access to these habitats for microbial diversity study. Due to nutrient limitation, subsurface microbiomes adapt, potentially producing biotechnologically important biomolecules. Planctomycetota, known for possessing about 20 to 45% of protein-coding genes of unknown function, may be relevant in this context. A percolate water sample from the weathered bedrock of the Hainich Critical Zone Exploratory (CZE; Thuringia, Germany) was processed to enrich planctomycetes, leading to the isolation of an uncharacterized Isosphaeraceae member, strain EP7^T. Strain EP7^T forms round, pink colonies, and spherical, non-motile cells that divide asymmetrically by budding. It grows between 10 and 24 °C and over a range of pH 5 to pH 10. Its genome size is 7.2 Mbp, and its DNA G + C content is 66.7%. Polyphasic characterization justifies the assignment of strain EP7^T to a novel species within a novel genus. We introduce the name Kueselia aquadivae for the novel taxon with strain EP7^T as the type strain of the novel species. Strain EP7^T represents the first Isosphaeraceae member isolated from vadose zone percolate water.

Keywords: Isosphaeraceae; Planctomycetota; Aeration zone; Critical zone; Drainage collectors; Geomicrobiology.

Fig. 1

Location of the sampling site. (A) Location of the Hainich CZE in Thuringia, Germany (indicated with the pin needle). (B) Isolation of strain EP7^T from a sample from the Drainage Collector ADI-DC-TK-01 (TK-1) in a mixed beech forest area in the Hainich CZE, maintained by the Collaborative Research Center AquaDiva. The maps were created using the ArcGis online function MapViewer provided by Esri (https://www.arcgis.com/home/webmap/viewer.html, version 02/2024, accession date: 14/06/2024).

Fig. 2

Maximum likelihood phylogenetic trees displaying the phylogenetic position of the novel strain isolated from the undisturbed subsurface. Maximum likelihood phylogenetic trees based on 16S rRNA gene sequences (A) and MLSA (B) were computed based on the current members of the phylum Planctomycetota (A) or the current members of the family Isosphaeraceae (B) (+ outgroups as given in the Material and methods section). Bootstrap values after 1000 re-sampling (A) or 500 re-samplings (B) are given at the nodes (in %). The trees were visualized using iTOL v6. The scale bar indicates the number of substitutions per nucleotide (A) or amino acid position (B).

Fig. 3

Analysis of phylogenetic markers. Strain EP7^T and its current closest relatives are displayed with the values of respective phylogenetic markers including 16S rRNA gene sequence similarity (16S), average amino acid identity (AAI), average nucleotide identity (ANI), percentage of conserved proteins (POCP) and similarity of a 1300 bp partial rpoB sequence. The genus/species threshold for each genetic marker is shown in the upper right corner. n.d. not determined (no genome sequence available). *The genus threshold for rpoB was determined for members of the families Planctomycetaceae and Pirellulaceae and might not be applicable for the other orders in the class Planctomycetia.

Fig. 4

Determination of temperature and pH range and optimum. Strain EP7^T grows over a range of 10 to 24 °C with an optimum between 18 to 21 °C and between pH 6.0 and 9.0 with an optimum at 7.0 to 8.0.

Fig. 5

Morphological features and cell division of strain EP7^T. (A) Phase contrast microscopy reveals cells of strain EP7^T to be spherical and to appear as single cells, pairs of two, or small chains. The scale bar represents 2 μm. (B) Cells of strain EP7^T have a mean length of 2.4 μm and a mean width of 2.2 μm representing an almost spherical shape. (C) Phase contrast time-lapse microscopy of strain EP7^T showing cell division via asymmetric cell division (“polar budding"). Scale bars represent 2 μm.

Fig. 6

Strain EP7^T takes up the FITC-labelled model polysaccharide dextran. Structured illumination microscopy of strain EP7^T cells incubated with DAPI (DNA stain), FITC-labelled dextran, and Synaptored (membrane stain). Fluorescence signals of the FITC-labelled dextran (green) co-localize with the fluorescence signal of the membrane stain (red) indicating an uptake of the polysaccharide into the periplasmic space. The DNA appears as condensed foci and is located in the cytoplasm next to the cytoplasmic membrane and periplasmic invaginations. Scale bars represent 2 μm.