Exometabolite dynamics over stationary phase reveal strain-specific responses to nutrient limitation

Abstract

AbstractMicrobial exponential growth is expected to occur infrequently outside of the laboratory, in the environment. Instead, resource-limited conditions impose non-growth states for microbes. However, non-growth states are uncharacterized for the majority of environmental bacteria, especially in regard to exometabolite production. To investigate exometabolite production in response to nutrient limitation, we compared exometabolites produced over time in stationary phase across three environmental bacteria: Burkholderia thailandensis E264 (ATCC 700388), Chromobacterium violaceum ATCC 31532, and Pseudomonas syringae pathovar tomato DC3000 (ATCC BAA-871). We grew each strain in monoculture and investigated exometabolite dynamics over time from mid-exponential to stationary phase. We focused on exometabolites that were released into the media and accumulated over 45 hours, including approximately 20 hours of stationary phase. In concert, we analyzed transcripts (RNAseq) to inform interpretation of exometabolite output. We found that a majority of exometabolites released under these conditions were strain-specific. A subset of identified exometabolites were involved in both central and secondary metabolism. Transcript analysis supported that exometabolites were released from intact cells, as various transporters were either upregulated or consistently expressed. Interestingly, we found that all strains released succinate, and that each strain re-routed their metabolic pathways involved in succinate production during stationary phase. Overall, these results show that non-growth states can also be metabolically active and dynamic. Furthermore, they show that environmental bacteria have the capability to transform a resource-limited extracellular environment into a rich chemical milieu. This work has implications for understanding microbial community interactions via exometabolites, and within resource-limited environments.ImportanceNon-growth states are common for bacteria that live in resource-limited environments, and yet these states remain largely uncharacterized in cellular metabolism and metabolite output. Here, we investigated and compared stationary phase exometabolites and RNA transcripts for each of three environmental bacterial strains. We observed that diverse exometabolites were produced and that they collectively exhibited clear and directional dynamics over time. Additionally, each bacteria strain had a characteristic exometabolite profile and dynamic. This work affirms that stationary phase is not at all “stationary” for these bacteria, and sets the stage for understanding how individual metabolisms support interspecies interactions in resource-limited environments.