High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits

Moonmilkare cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothemsappealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilkdeposits of the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. Actinobacteriawas the most common phylumafter Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units(OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia(9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinkedto the culturable Streptomyces strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three studied collection points, while 34% were exclusive to one deposit, indicating that distinct speleothemshost their own population, despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilkdeposits should result in the isolation of unique and novel Actinobacteria. These speleothemsalso host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.