CO2 exacerbates oxygen toxicity

CO2 exacerbates oxygen toxicity Supplementary information

Benjamin Ezraty, Maïalène Chabalier, Adrien Ducret, Etienne Maisonneuve and Sam Dukan

Aix Marseille Université - Laboratoire de Chimie Bactérienne (UPR 9043) - Institut de Microbiologie de la Méditerranée (IFR88) - CNRS, 31, Chemin Joseph Aiguier, 13402, Marseille, France.

Corresponding author: Sam Dukan, LCB CNRS, 31, Chemin Joseph Aiguier, 13402, Marseille, France, Tel: 0033 491 16 46 01, e-mail: [email protected]

Supplementary information 1

Determination of a sufficient atmospheric CO2 level concentration required for E. coli growth. As CO2 is required for normal microbial growth, we first determined the CO2 concentration sufficient for optimal E. coli growth. We used both time-lapse experiments to follow the first cell divisions and growth experiments on LB agar plates to follow later cell divisions. In the absence of CO2 (0 ppm), cell growth was initially slower and ceased after about seven divisions (Figure S1A and S1B), confirming the requirement of CO2 for E. coli growth (Walker, 1932; required for fatty acid biosynthesis and arginine biosynthesis, for example). We established that 40 ppm was a sufficiently high atmospheric CO2 concentration for optimal growth. Below this concentration, growth was slower. Above this concentration and at concentrations of up to 1,000 ppm, cell division rates and colony formation rates on plates were similar (Figure S1A and S1B, see also video in supplementary information). CO2 is thus required for growth but is not toxic per se at concentrations of up to 1,000 ppm.

Figure S1. E. coli growth at various atmospheric CO2 levels. E. coli growth was similar at all atmospheric CO2 concentrations tested in the range of 40 to 1,000 ppm. E. coli growth at various

atmospheric CO2 concentrations was assessed by time-lapse experiments and the monitoring of clones on LB agar plates. (A) Still frames (phase contrast) of the development of a microcolony from a single cell. E. coli (MG1655) was cultured on an LB agar (1.2%) matrix (agar-pad) previously equilibrated at the working atmospheric CO2 concentration for two hours. The system was then mounted in a sealedflow chamber allowing constant aeration with the working atmospheric CO2 level (0, 40, 300 and 1,000 ppm) over a period of 325 minutes. Scale bar 10 µm. (B) Growth of an E. coli colony on an LB agar-plate at various atmospheric CO2 concentrations (0, 40, 300 and 1,000 ppm) over a period of 18 hours. Scale bar: 1 mm.


Movie S1. The growth of an E. coli (MG1655) microcolony at various atmospheric CO2 concentrations. Cells were placed on the microscope coverslip and covered with a thin layer (1 mm thick) of semisolid LB agar (1.2%) matrix (agar-pad), previously equilibrated at the working atmospheric CO2 concentration for two hours. The coverslip and agar-pad were then mounted in a sealed-flow chamber allowing constant aeration with the working atmospheric CO2 concentration. Flow chambers were incubated in a controlled-temperature (Solent-Scientific, United Kingdom) automated microscope (Nikon TE2000-E-PFS, Nikon, France) at 37±1°C for up to six hours. For each experiment, ten fields, each containing at least one cell, were identified manually and stored for timelapse experiments. Images of each field were taken, at five-minute intervals, with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 40x/0.75 DLL “Plan-Apochromat”. Timelapse experiments, digital analysis and image processing were carried out with Metamorph version 7.5 (Molecular Devices France, France).

This film shows the growth of an E. coli microcolony over 325 min (33 frames), condensed into 11 s. The data obtained from this microcolony were used for Figure 1.


Figure S2. Atmospheric CO2 concentration (40 – 1,000 ppm) has no effect on E. coli metabolism E. coli (MG1655) cells were grown under an atmosphere containing 40 or 1,000 ppm CO2 to an OD600 of 0.5. Superoxide dismutase (Beauchamp & Fridovich, 1971), glucose-6-phosphate dehydrogenase (Fraenket & Levisohn, 1967), glyceraldehyde-3-phosphate dehydrogenase (Huang et al, 2000), malate dehydrogenase (Courtright & Henning, 1970) activities and the turnover of bulk proteins (Geuskens et al, 1992) were thus estimated with cells cultured in atmospheres containing 40 or 1,000 ppm CO2.


Cell staining for intracellular pH measurement. All experiments were carried out at 37°C, in a dark room. A stock solution of 1 mM BCECF-AM (Molecular Probes) in DMSO was stored in the dark at 20°C. Cells were incubated at 37°C, on a rotary shaker, with 500 µM BCECF-AM, for incubation times of 15 min, at CO2 concentrations of 40 or 1,000 ppm. Cells were immobilized in a commercial flow cell (µ-Slide VI, Ibidi) and rinsed twice in the initial medium. The carboxylic ionophore nigericin was used for intracellular pH calibration in vivo, as described elsewhere (Negulescu et al, 1990; Corvini et al, 2000). Nigericin was dissolved in absolute ethanol at a final concentration of 1 mM. The cells used for the establishment of calibration curves were stained and immobilized in the flow chamber, as described above. After deposition, cells were washed with high [K+] buffer at various pH values (5-8). The high [K+] buffer used was Hanks Balanced Salt Solution: 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3 and 5 mM glucose. Buffer solutions were filtered before use through filters with 0.22 µm pores and were then stored at 4°C. Nigericin was added to a final concentration of 10 µM and the cells were incubated for 10 min. Cells were observed on an epifluorescence microscope with x 1000 magnification. The excitation filter was the same for both modules (480/15 nm), but two different emission filters were used: 535/20 (green) and 610/75 (red).

Figure S3. Atmospheric CO2 concentration (40 – 1,000 ppm) has no effect on E. coli intracellular pH (A) The intracellular pH of cells harvested at an OD600 of 0.2 and growing in concentrations of 40 or 1,000 ppm CO2 (B) Calibration curve of E. coli. The calibration curve was determined on 100 individual cells for each intracellular pH tested. (C) Representative result for stained cells with excitation at 488 nm and observation of 535 and 610 nm.


Figure S4. Atmospheric CO2 concentration (40 – 1,000 ppm) has no effect on the pH of the LB broth supporting WT E. coli growth. LB broth pH was measured at various times after the removal of the cells (E. coli MG1655) by centrifugation.


Figure S5. E. coli growth at various atmospheric CO2 concentrations after a shift from anaerobic to aerobic conditions. WT E. coli (MG1655) was cultured in anaerobiosis and shifted to aerobiosis under atmospheres containing various concentrations of CO2 (40, 300, and 1,000 ppm), as described in the methods section.


Figure S6. No effect of atmospheric CO2 concentration on Hpx- dps strain by modulation of ROS steady state. Hpx- dps strain was cultured in anaerobiosis containing 40 ppm of CO2 as described in the methods section and shifted to atmospheres containing 40 or 1,000 ppm CO2 in anaerobiosis (A) or in aerobiosis, and in the presence of catalase (5,000 U/ml) (B) or DMPO (100 µM) (C). Representative results are presented in the figure and each analysis was repeated three times to confirm reproducibility.


8-oxo-guanine detection by immunofluorescence. We dispensed the pellet obtained from 30 µl of cell suspension washed, resuspended and diluted 1:10 in phosphate buffer (0.05 M, pH 7.4) into poly L-lysine-coated eight-well slides and incubated the slides for 10 minutes at room temperature. We then added 30 µl of 4% formaldehyde and incubated the slides for 15 minutes. Wells were then washed three times with phosphate buffer (0.05 M, pH 7.4) and once with GTE (50 mM glucose, 20 mM Tris-HCl pH 7.5, 10 mM EDTA). Cells were treated with 30 µl of lysozyme (0.2 µg/ml in GTE) for 10 minutes at 37°C and were then washed five times with phosphate-buffered saline (PBS; 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4) and once with ethanol (50 to 80 and 100%). The wells were rehydrated with 30 µl of Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.4 and 150 mM NaCl) supplemented with 2% BSA, 0.1% Tween (TBST-BSA) and incubated for 30 minutes. We added 30 µl of goat anti-8-OHdG polyclonal antibody (Millipore) diluted 1:500 in TBST-BSA to each well, and incubated the slides for 2 h at room temperature. Each well was then washed five times with TBST-BSA. We added 30 µl of Alexa fluor 546-conjugated anti-goat secondary antibody (Invitrogen) diluted 1:500 in TBST-BSA to each well and incubated the slides for 1 h at room temperature. Wells were washed five times, and 3 µl of Citifluor (Biovalley) was added.

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