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 long-term 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.

FIG 1

(A) Survival dynamics of PFM2 in tubes and flasks. Shown are growth curves of PFM2 in 5 ml in tubes (squares) and 12.5 ml in flasks (circles). Average data shown are from a representative experiment of three biological replicates. Error bars represent standard deviations. (B) pH of the same samples as in panel A.

FIG 2

Survival dynamics of PFM2 in cultures of different volumes. Growth curves (A) and pH (B) of PFM2 incubated in 2 ml (squares), 5 ml (circles), or 12.5 ml (triangles) of media in test tubes are shown. Growth curves (C) and pH (D) of PFM2 cultured in 5 ml (squares), 12.5 ml (circles), 25 ml (triangles), or 50 ml (diamonds) of media in flasks are also shown. Data are the averages of three biological replicates; error bars represent standard deviations.

FIG 3

Survival dynamics of PFM2 in anaerobic cultures. Growth curves (A) and pH (B) of PFM2 in anaerobic cultures in flasks are shown for the following volumes: 5 ml (squares), 12.5 ml (circles), 25 ml (triangles), and 50 ml (diamonds). Data are the averages of three biological replicates; error bars represent standard deviations.

FIG 4

Spontaneous rifampin resistance mutations in different culture volumes. Median values (n = 15) for the number of rifampin-resistant mutants in different culture conditions are shown per 10⁷ CFU/ml for 5 ml in tubes (hatched bar), 5 ml in flasks (black bar), 12.5 ml in flasks (white bar), 25 ml in flasks (dark gray bar), and 50 ml in flasks (light gray bar).

FIG 5

Expression of P_oxyR in different culture volumes in flasks. β-Galactosidase assays were performed on PFM2/pAQ23 (SF2562) during long-term growth in 5 ml (black bar), 12.5 ml (white bar), 25 ml (dark gray bar), and 50 ml (light gray bar). Bars represent average modified Miller units (see Materials and Methods) of duplicate samples. Error bars represent standard deviations. *, P < 0.05 using Student's t test.

FIG 6

ELISA showing differences in glycation after overnight incubation using an anti-CML antibody. Assays were performed on total cellular protein from PFM2 during long-term growth in 5-ml (black bar), 12.5-ml (white bar), 25-ml (dark gray bar), or 50-ml (light gray bar) flask cultures. Bars represent average glycation/mg of protein of duplicate samples. Error bars represent standard deviations. *, P < 0.05 using Student's t test.

FIG 7

Carnosine partially rescues cells from death phase. Growth without (solid lines) or with (dotted lines) 50 mg/ml of carnosine in 5-ml (A; squares), 12.5-ml (B; circles), 25-ml (C; triangles), or 50-ml (D; diamonds) cultures in flasks is shown. Lines represent average of four biological replicates over two separate experiments; error bars represent standard deviations.