RESUMEN
The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms. IMPORTANCE Bacteria are capable of producing and secreting a rich diversity of specialized metabolites. Yet, much of their exometabolome remains hidden due to challenges associated with eliciting specialized metabolite production, labor-intensive sample preparation, and time-consuming analysis techniques. Using our versatile three-dimensional (3D)-printed culturing platform, SubTap, we demonstrate that rapid exometabolomic data collection from a diverse set of environmental bacteria is feasible. We optimized our platform by surveying Streptomyces isolated from soil on a variety of media types to assess viability, degree of specialized metabolite production, and compatibility with downstream LESA-DIMS analysis. Ultimately, this will enable data-rich experimentation, allowing for a better understanding of bacterial exometabolomes.
Asunto(s)
Productos Biológicos , Streptomyces , Espectrometría de Masas/métodos , Suelo/química , Productos Biológicos/químicaRESUMEN
Communication within the microbiome occurs through an immense diversity of small molecules. Capturing these microbial interactions is a significant challenge due to the complexity of the exometabolome and its sensitivity to environmental stimuli. Traditional methods for acquiring exometabolomic data from interacting microorganisms are limited by their low throughput or lack of sampling depth. To address this challenge, we introduce subtapping (short for substrate tapping), a technique for tapping into extracellular metabolites that are being transferred through the growth substrate during coculture. High-throughput subtapping is made possible by a new coculturing platform, named SubTap, that we engineered to resemble a 96-well plate. The three-dimensional (3D) printed SubTap platform captures the exometabolome in an agar compartment that connects physically separated growth chambers, which permits cell growth without competition for space. We show how SubTap facilitates replicable and quick detection of exometabolites via direct infusion mass spectrometry analysis. Using bacterial isolates from the soil, we apply SubTap to characterize the effects of growth medium, growth duration, and mixed versus unmixed coculturing on the exometabolome. Finally, we demonstrate SubTap's versatility by interrogating microbial interactions in multicultures with up to four strains. IMPORTANCE Improvements in experimental techniques and instrumentation have led to the discovery that the microbiome plays an essential role in human and environmental health. Nevertheless, there remain major impediments to conducting large-scale interrogations of the microbiome in a high-throughput manner, particularly in the field of exometabolomics. Existing methods to coculture microorganisms and interrogate their interactions are labor-intensive and low throughput. This inspired us to develop a solution for coculturing that was (i) open source, (ii) inexpensive, (iii) scalable, (iv) customizable, and (v) compatible with existing mass spectrometry instrumentation. Here, we present SubTap-a 3D printed coculturing platform that permits tapping directly into the growth substrate between physically separated, but interconnected, growth compartments. SubTap allows multiculture (with up to four distinct growth compartments) in spatially mixed or unmixed configurations and enables repeatable results with mass spectrometry, as shown by our validation with known compounds and cultures of one to four organisms.