A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria

Abstract

Exometabolomics is an approach to assess how microorganisms alter, or react to their environments through the depletion and production of metabolites. It allows the examination of how soil microbes transform the small molecule metabolites within their environment, which can be used to study resource competition and cross-feeding. This approach is most powerful when used with defined media that enable tracking of all metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse soil bacteria but also is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 108 of the 110 phylogenetically diverse (spanning 36 different families) soil bacterial isolates tested and all of its metabolites were trackable through LC–MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for growing and characterizing diverse microbial isolates and communities.

Reproducible growth ofBrachypodium distachyonin fabricated ecosystems (EcoFAB 2.0) reveals that nitrogen form and starvation modulate root exudation

“…Synthesis of previous observations and our exometabolomics, microbial, and root growth data (Fig. 3, 4, and 5) suggest that exogenous shikimic acid can promote root growth, serve as substrates to support the growth of soil bacteria, and thus shape microbiome assembly in the rhizosphere (3,6,20,48).…”

“…This is consistent with other studies showing that rhizosphere bacteria prefer to consume shikimic acid alongside other organic acids (nicotinic, salicylic, cinnamic, and indole-3-acetic) exuded by roots of grass Avena barbata ( 3 ). Moreover, shikimic acid was shown to be a highly consumed substrate by diverse phylogeny of soil isolates in the recently developed Northen Lab Defined Medium (NLDM) ( 48 ). The shikimic acid promoted the growth of the roots only under iN+ conditions relative to a control ( Fig.…”

Substrate Utilization and Competitive Interactions Among Soil Bacteria Vary With Life-History Strategies

“…Recent advances in mass spectrometry-based metabolomics help fill this gap by enabling comprehensive analyses of chemically diverse samples ( Bauermeister et al, 2022 ). By profiling changes of extracellular metabolites (“exometabolites”) in medium before and after cultivation (i.e., control vs. “spent” medium), we can characterize substrates that are preferentially depleted as well as metabolites that are secreted by individuals or communities of microorganisms ( Silva and Northen, 2015 ; de Raad et al, 2022 ). Using an exometabolomics approach, we previously demonstrated that sympatric bacteria isolated from biocrusts have divergent substrate preferences ( Baran et al, 2015 ).…”

Runella salmonicolor sp. nov. and Dyella lutea sp. nov., isolated from paddy field soil

“…A soil sample was suspended in 5 ml 0.85 % NaCl and rotated for 30 min in a 50 ml conical tube [17]. Then, 200 µl serial dilution (10 −1 , 10 −2 , 10 −3 and 10 −4 ) was performed, and 100 µl of the diluted solution were suctioned and spread on Reasoner’s 2A (R2A; MB cell) agar plates [18]. The plates were incubated at 25 °C for 3 days.…”