Abstract
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.
Highlights
Bioprospecting for natural compounds from microorganisms dwelling in poorly explored and extreme environments has gained renewed interest [1,2], boosted by the need to fight resistance to compounds currently used as antimicrobials, herbicides, antivirals, and anticancer agents [3].Caves, despite being highly oligotrophic environmental niches, support rich and diverse microbial life, a phenomenon called “the Paradox of the Plankton”, which suggests that, in spite of limited nutrient resources, an unexpectedly wide range of species coexist [4]
The moonmilk suspension was inoculated in the two media that showed the most contrasting isolation efficiency in our previous screening [12]—(i) the International Streptomyces Project (ISP) medium number 5 (ISP5) medium in which no colonies could be observed; and (ii) the starch nitrate (SN) medium, which gave the highest number of colony forming units (CFUs)
In order to reduce the formation of highly toxic reactive oxygen species (ROS) that might be generated during autoclaving through the interaction of components of the media, phosphate and agar [22,23], ISP5 and SN were prepared in two different manners—(i) either with all of the components autoclaved together or (ii) with agar and phosphate autoclaved separately from all of the other components
Summary
Bioprospecting for natural compounds from microorganisms dwelling in poorly explored and extreme environments has gained renewed interest [1,2], boosted by the need to fight resistance to compounds currently used as antimicrobials, herbicides, antivirals, and anticancer agents [3].Caves, despite being highly oligotrophic environmental niches, support rich and diverse microbial life, a phenomenon called “the Paradox of the Plankton”, which suggests that, in spite of limited nutrient resources, an unexpectedly wide range of species coexist [4]. High-throughput sequencing (HTS) of DNA extracted from the same moonmilk deposits demonstrated that we failed to isolate representatives of at least 46 additional actinobacterial genera [13] Such an outcome might be related to the fact that Streptomyces are adapted to use a wide range of nutrients and grow effectively on many selective media, and to the fact that their growth is faster in comparison to that of other actinobacterial genera. For this reason, non-streptomycetes, which are more challenging to isolate in pure cultures, are regarded as rare Actinobacteria. The 78 culturable Streptomyces strains isolated in our previous screening [11] constitute a minor fraction of those dwelling in the studied moonmilk deposits, because we only recovered relatives of 5 out of the 19 operational taxonomic units (OTUs) affiliated with the Streptomyces genus [13], suggesting that this environment hosts hard-to-culture Streptomyces species that are potentially highly diverged from their soil-dwelling counterparts
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