Actinobacterial Spatial Diversity in Moonmilk

Marta Maciejewska1, Magdalena Całusińska2, Luc Cornet3, Delphine Adam1, Igor S. Pessi1, Sandrine 5 Malchair4, Philippe Delfosse2, Denis Baurain3, Hazel H. Barton5, Monique Carnol4, and Sébastien 6 Rigali1* 7 8 1 InBioS Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Liège, Belgium 9 2Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, 10 Belvaux, Luxembourg 11 3 InBioS PhytoSYSTEMS, Eukaryotic Phylogenomics, University of Liège, Liège, Belgium 12 4 InBioS Plant and Microbial Ecology, Botany B22, University of Liège, Liège, Belgium 13 5 Department of Biology, University of Akron, Akron, Ohio, United States of America 14 * Correspondence: [email protected]; Tel.: + 32 4 366 98 30 15 16 Abstract: Moonmilk are cave carbonate deposits that host a rich microbiome including antibiotic17 producing Actinobacteria making these speleothems appealing for bioprospecting. Here we 18 investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of 19 the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. 20 Actinobacteria was the most common phylum after Proteobacteria, ranging from 9 to 23% of the total 21 bacterial population. Next to actinobacterial OTUs attributed to uncultured organisms at the genus 22 level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and 23 Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of absolute number of sequences. 24 Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of OTUs unlinked to the 25 culturable Streptomyces strains previously isolated from the same deposits. 43% of OTUs were shared 26 between the three studied collection points while 34% were exclusive to one deposit indicating that 27 distinct speleothems host their own population despite their nearby localization. This important 28 spatial diversity suggests that prospecting within different moonmilk deposits should result in the 29 isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non30 streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for 31 isolating rare Actinobacteria. 32