Culture Volume and Vessel Affect Long-Term Survival, Mutation Frequency, and Oxidative Stress of Escherichia coli

Abstract

Bacteria such as Escherichia coli are frequently studied during exponential-and stationary-phase growth. However, many strains can survive in long-term stationary phase (LTSP), without the addition of nutrients, from days to several years. During LTSP, cells experience a variety of stressors, including reactive oxidative species, nutrient depletion, and metabolic toxin buildup, that lead to physiological responses and changes in genetic stability. In this study, we monitored survival during LTSP, as well as reporters of genetic and physiological change, to determine how the physical environment affects E. coli during longterm batch culture. We demonstrate differences in yield during LTSP in cells incubated in LB medium in test tubes versus Erlenmeyer flasks, as well as growth in different volumes of medium. We determined that these differences are only partially due to differences in oxygen levels by incubating the cells in different volumes of media under anaerobic conditions. Since we hypothesized that differences in long-term survival are the result of changes in physiological outputs during the late log and early stationary phases, we monitored alkalization, mutation frequency, oxidative stress response, and glycation. Although initial cell yields are essentially equivalent under each condition tested, physiological responses vary greatly in response to culture environment. Incubation in lower-volume cultures leads to higher oxyR expression but lower mutation frequency and glycation levels, whereas incubation in high-volume cultures has the opposite effect. We show here that even under commonly used experimental conditions that are frequently treated as equivalent, the stresses experienced by cells can differ greatly, suggesting that culture vessel and incubation conditions should be carefully considered in the planning or analysis of experiments.

Antibiotics induce redox-related physiological alterations as part of their lethality

“…For example, depending on the specific drug being assayed, we observe between 1-4 logs of killing, which can be perturbed by extrinsic factors such as the level of aeration and availability of terminal electron acceptors. Interesting recent work has illustrated that phenotypes such as cell death, mutagenesis, oxidative stress, and related activation of OxyR can all be reduced, depending on the method of culturing (101). These findings are consistent with a recent commentary on the current ROS debate (93), which suggested that differences in experimental conditions may help explain the incongruent conclusions regarding the ROS hypothesis reached by others based on the absence or minimal extent of such phenotypes (49)(50)(51).…”

Rich Medium Composition Affects Escherichia coli Survival, Glycation, and Mutation Frequency during Long-Term Batch Culture

“…Monitoring cell growth, survival, culture pH, and mutation frequency. To monitor cell growth and survival, viable cell counts were determined by serial dilution of cells sampled periodically from the cultures, followed by plating on LB agar (2,25). The limit of detection in all experiments was Ͼ1,000 CFU/ml (25).…”

“…Enzyme-linked immunosorbent assays (ELISAs) were performed as previously described (2). Briefly, 50 l of whole-cell lysate (protein concentration, 0.5 to 1.0 mg/ml) were loaded into standard polystyrene 96-well plates (Corning, Inc.) with 100 l sodium carbonate binding buffer and incubated overnight at 4°C.…”

“…Whole-cell protein was isolated as previously described (2,10). Briefly, 5-ml cultures of PFM2, grown in different media, were incubated for 1 to 3 days.…”

“…volume (2). While it is clear that glycation, culture pH, and mutation frequency contribute to death phase and survival in the LTSP, the specific relationships between these phenotypes are not well understood.…”