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ABSTRACT Bacteria need to be isolated in pure culture to gain access to the wide array of interesting functions they conceal. This is challenging because they do not live in isolation but instead in communities where they actively communicate and interact with each other. In this study, we aimed to increase the culturability of soil bacteria using three different signaling molecules: N-(3-oxohexanoyl)-L-homoserine lactone, N-octanoyl-L-homoserine lactone, and 3',5'-cyclic adenosine monophosphate (cAMP). The signals were added individually to soils suspended in PBS buffer to a final concentration of 5 µM. Soil suspensions were agitated for 24 hours, after which they were serially diluted and plated on tenfold-diluted Reasoner’s 2A agar that either contained or did not contain the same signaling molecule used during the extraction step. DNA was isolated from both soil suspensions and grown cultures and, after a high-throughput amplicon sequencing, differences in bacterial abundances and diversity were determined across treatments. To further explain the action of the signaling molecules on the treated soil communities, their metagenomic functions were predicted using the software PICRUSt2. N-octanoyl-L-homoserine lactone was found to increase the diversity observed on solid media, while N-(3-oxohexanoyl)-L-homoserine lactone and cAMP were not. Potentially novel isolates aided by the signaling molecules were affiliated with the genera Pseudomonas and Nocardioides. IMPORTANCE Microorganisms are a repository of interesting metabolites and functions. Therefore, accessing them is an important exercise for advancing not only basic questions about their physiology but also to advance technological applications. In this sense, increasing the culturability of environmental microorganisms remains an important endeavor for modern microbiology. Because microorganisms do not live in isolation in their environments, molecules can be added to the cultivation strategies to “inform them” that they are present in growth-permissive environmental conditions. Signaling molecules such as acyl-homoserine lactones and 3',5'-cyclic adenosine monophosphate belong to the plethora of molecules used by bacteria to communicate with each other in a phenomenon called quorum sensing. Therefore, including quorum sensing molecules can be an incentive for microorganisms, specifically soil bacteria, to increase their numbers on solid media.

ABSTRACT Balneotherapeutic water springs, such as those with thermal, saline, sulfur, or any other characteristics, have recently been the subject of phylogenetic studies with a closer focus on the description and/or isolation of phylogenetically novel or biotechnologically interesting microorganisms. Generally, however, most such microorganisms are rarely obtained in pure culture or are even, for now, unculturable under laboratory conditions. In this culture-dependent study of radioactive water springs of Jáchymov (Joachimstahl), Czech Republic, we investigated a combination of classical cultivation approaches with those imitating sampling source conditions. Using these environmentally relevant cultivation approaches, over 1,000 pure cultures were successfully isolated from 4 radioactive springs. Subsequent dereplication yielded 121 unique taxonomic units spanning 44 genera and 9 taxonomic classes, ~10% of which were identified as hitherto undescribed taxa. Genomes of the latter were sequenced and analyzed, with a special focus on endogenous defense systems to withstand oxidative stress and aid in radiotolerance. Due to their origin from radioactive waters, we determined the resistance of the isolates to oxidative stress. Most of the isolates were more resistant to menadione than the model strain Deinococcus radiodurans DSM 20539T. Moreover, isolates of the Deinococcacecae, Micrococcaceae, Bacillaceae, Moraxellaceae, and Pseudomonadaceae families even exhibited higher resistance in the presence of hydrogen peroxide. In summary, our culturomic analysis shows that subsurface water springs contain diverse bacterial populations, including as-yet-undescribed taxa and strains with promising biotechnological potential. Furthermore, this study suggests that environmentally relevant cultivation techniques increase the efficiency of cultivation, thus enhancing the chance of isolating hitherto uncultured microorganisms. IMPORTANCE The mine Svornost in Jáchymov (Joachimstahl), Czech Republic is a former silver-uranium mine and the world’s first and for a long time only radium mine, nowadays the deepest mine devoted to the extraction of water which is saturated with radon and has therapeutic benefits given its chemical properties. This healing water, which is approximately 13 thousand years old, is used under medical supervision for the treatment of patients with neurological and rheumatic disorders. Our culturomic approach using low concentrations of growth substrates or the environmental matrix itself (i.e., water filtrate) in culturing media combined with prolonged cultivation time resulted in the isolation of a broad spectrum of microorganisms from 4 radioactive springs of Jáchymov which are phylogenetically novel and/or bear various adaptive or coping mechanisms to thrive under selective pressure and can thus provide a wide spectrum of capabilities potentially exploitable in diverse scientific, biotechnological, or medical disciplines.