A Thousand Meters Deep: Vertical Profiling of the Subterranean Microbiome of Gourgouthakas Cave

Paragkamian, S.; Christakis, C. A.; Michalopoulou, V. A.; Plakogiannaki, M.; Soultatos, S.; Arapitsas, N. P.; Vaxevanopoulos, M.; Sotiriadis, Y.; Pennos, C.; Markakis, E. A.; Sarris, P. F.

doi:10.64898/2026.03.19.712943

bioRxiv · 10.64898/2026.03.19.712943

A Thousand Meters Deep: Vertical Profiling of the Subterranean Microbiome of Gourgouthakas Cave

Paragkamian, S.·Christakis, C. A.·Michalopoulou, V. A.·Plakogiannaki, M.·Soultatos, S.·Arapitsas, N. P.·Vaxevanopoulos, M.·Sotiriadis, Y.·Pennos, C.·Markakis, E. A.·Sarris, P. F.

View original source ↗Export metadata ↓

Abstract

IntroductionCaves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we conducted a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the worlds deepest vertical systems, which had remained untouched by humans for 19 years. MethodsWe performed a high-resolution vertical profiling of the caves microbes by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation on various media and at different temperatures, we established a biobank of 820 bacterial isolates. ResultsTaxonomic identification of a 374-isolate subset revealed a diverse community spanning 35 genera and 4 phyla, dominated by Pseudomonas, Aquipseudomonas, Bacillus, and Stenotrophomonas. Beyond characterizing this taxonomic diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum, Verticillium dahliae, and Phytophthora nicotianae, uncovered a notable group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria (Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs), over half of which showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Pangenomic analysis of Streptomyces revealed 1,497 unique gene clusters. Finally, ex vivo trials showed that the Aquipseudomonas paracarnis (formerly Pseudomonas sp.) isolate SRL917 significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. DiscussionCollectively, our results demonstrate that deep karstic systems are not merely geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Paragkamian, S., Christakis, C. A., Michalopoulou, V. A., Plakogiannaki, M., Soultatos, S., Arapitsas, N. P., Vaxevanopoulos, M., Sotiriadis, Y., Pennos, C., Markakis, E. A., Sarris, P. F.. 2026-03-19. A Thousand Meters Deep: Vertical Profiling of the Subterranean Microbiome of Gourgouthakas Cave. https://doi.org/10.64898/2026.03.19.712943

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗

bioRxiv2026

The iron-binding siderophore enterobactin is required for the response of multi-drug resistant Klebsiella pneumoniae to zinc limitation

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

microbiology↗

bioRxiv2026

A microbiota-derived protease links phage susceptibility to host epithelial responses

Bacteriophages are major ecological drivers of gut microbial ecology, yet whether bacterial mechanisms that determine phage susceptibility have consequences for the mammalian host remains poorly understood. Here, we identify dipeptidyl peptidase 11 (Dpp11a), the predominant active serine protease of the prevalent gut commensal Phocaeicola vulgatus, as an unexpected bacterial defence factor. Dpp11a protects against environmental proteases and confers resistance to bacteriophage infection. Metatranscriptomic analyses further reveal increased expression of both dpp11a and P. vulgatus-associated phage transcripts in ulcerative colitis stool samples, indicating that both components of this interaction are transcriptionally active in disease-associated human microbiomes. Using the microfluidic gut-on-a-chip co-culture model HuMiX, we show that the absence of Dpp11 is accompanied by altered epithelial tight-junction remodelling during phage-bacterial infection. Together, our findings reveal that the consequences of bacterial phage defence can extend beyond phage-bacterium interactions to the mammalian epithelium.

microbiology↗