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

Abstract

15Microbial exponential growth is expected to occur infrequently outside of the 16 laboratory, in the environment. Instead, resource-limited conditions impose non-growth 17 states for microbes. However, non-growth states are uncharacterized for the majority of 18 environmental bacteria, especially in regard to exometabolite production. To investigate 19 exometabolite production in response to nutrient limitation, we compared 20 exometabolites produced over time in stationary phase across three environmental 21 bacteria: Burkholderia thailandensis E264 (ATCC 700388), Chromobacterium 22 violaceum ATCC 31532, and Pseudomonas syringae pathovar tomato DC3000 (ATCC 23 BAA-871). We grew each strain in monoculture and investigated exometabolite 24 dynamics over time from mid-exponential to stationary phase. We focused on 25 phase is not at all "stationary" for these bacteria, and sets the stage for understanding 49 how individual metabolisms support interspecies interactions in resource-limited 50 environments. 51 52 Keywords 53 Burkholderia thailandensis, Chromobacterium violaceum, Pseudomonas syringae, 54 secondary metabolism, RNAseq, mass spectrometry, metabolomics, persistence, non-55 growth state 56 57 Introduction 58Much of microbiology research in the laboratory is conducted with bacterial or 59 archaeal cells that are growing exponentially. However, it is estimated that 60% of 60 microbial biomass in the environment is in a non-growing state (1, 2). Various abiotic 61 and biotic stressors are known to induce non-growth states, but perhaps the most 62 common is resource limitation. Labile resources can be low in an environment, as 63 characteristic of the oligotrophic open ocean, or they can be available but inaccessible, 64 as typical in heterogeneous soil matrices. Thus, unlike most cultivated laboratory 65 strains, environmental microbes experience short periods of accessible resources 66 punctuated by long periods of famine (3, 4). 67 While Gram-positive bacteria can survive resource limitation through sporulation 68 (5), Gram-negative bacteria can persist in stationary phase without entry into a 69 specialized dormant cell structure (6). Instead, Gram-negative bacteria survive in 70 stationary phase by employing various stress response adaptations (7). Stress 71 4 response adaptations include changes to cell morphology, transcription, translation, and 72metabolism. Furthermore, in stationary phase, microbes can re-route metabolic 73 pathways to maintain essential components of the cell and the proton motive force (8). 74While these adaptations are thought to serve as survival mechanisms, the levels and 75 types of metabolic activities in stationary phase are not well understood for most 76 environmental microbes. 77It is known, however, that microbes can exhibit appreciable metabolic activity in 78 stationary phase (9). For example, entry into stationary phase resulted in prolonged 79 protein production in Escherichia coli despite that overall protein levels decreased (10). 80Metabolomic studies of E. coli in...