Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant Rasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing Streptomyces, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.

Cave moonmilk deposits host an abundant and diverse actinobacterial population that has a great potential for producing novel natural bioactive compounds. In our previous attempt to isolate culturable moonmilk-dwelling Actinobacteria, only Streptomyces species were recovered, whereas a metagenetic study of the same deposits revealed a complex actinobacterial community including 46 actinobacterial genera in addition to streptomycetes. In this work, we applied the rehydration-centrifugation method to lessen the occurrence of filamentous species and tested a series of strategies to achieve the isolation of hard-to-culture and rare Actinobacteria from the moonmilk deposits of the cave “Grotte des Collemboles”. From the “tips and tricks” that were tested, separate autoclaving of the components of the International Streptomyces Project (ISP) medium number 5 (ISP5) medium, prolonged incubation time, and dilution of the moonmilk suspension were found to most effectively improve colony forming units. Taxonomic analyses of the 40 isolates revealed new representatives of the Agromyces, Amycolatopsis, Kocuria, Micrococcus, Micromonospora, Nocardia, and Rhodococcus species, as well as additional new streptomycetes. The applied methodologies allowed the isolation of strains associated with both the least and most abundant moonmilk-dwelling actinobacterial operational taxonomic units. Finally, bioactivity screenings revealed that some isolates displayed high antibacterial activities, and genome mining uncovered a strong potential for the production of natural compounds.

Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that peptides/amino acids ammonification, and to a lesser extend ureolysis, could be privileged metabolic pathways participating in carbonate precipitation by increasing the pH of the bacterial environment. Additionally, in silico search for the genes involved in biomineralization processes including ureolysis, dissimilatory nitrate reduction to ammonia, active calcium ion transport, and reversible hydration of CO2 allowed to identify genetic predispositions for carbonate precipitation in Streptomyces. Finally, their biomineralization abilities were confirmed by environmental SEM, which allowed to visualize the formation of abundant mineral deposits under laboratory conditions. Overall, our study provides novel evidences that filamentous Actinobacteria could be key protagonists in the genesis of moonmilk through a wide spectrum of biomineralization processes.

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions. IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

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