Abstract
Using rpoS, tolC, ompF, and recA knockouts, we investigated their effect on the physiological response and lethality of ciprofloxacin in E. coli growing at different rates on glucose, succinate or acetate. We have shown that, regardless of the strain, the degree of changes in respiration, membrane potential, NAD+/NADH ratio, ATP and glutathione (GSH) strongly depends on the initial growth rate and the degree of its inhibition. The deletion of the regulator of the general stress response RpoS, although it influenced the expression of antioxidant genes, did not significantly affect the tolerance to ciprofloxacin at all growth rates. The mutant lacking TolC, which is a component of many E. coli efflux pumps, showed the same sensitivity to ciprofloxacin as the parent. The absence of porin OmpF slowed down the entry of ciprofloxacin into cells, prolonged growth and shifted the optimal bactericidal concentration towards higher values. Deficiency of RecA, a regulator of the SOS response, dramatically altered the late phase of the SOS response (SOS-dependent cell death), preventing respiratory inhibition and a drop in membrane potential. The recA mutation inverted GSH fluxes across the membrane and abolished ciprofloxacin-induced H2S production. All studied mutants showed an inverse linear relationship between logCFU ml−1 and the specific growth rate. Mutations shifted the plot of this dependence relative to the parental strain according to their significance for ciprofloxacin tolerance. The crucial role of the SOS system is confirmed by dramatic shift down of this plot in the recA mutant.
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This work was carried out in accordance with state assignment AAAA-A19-119112290009-1 and supported by grants from the Russian Foundation for Basic Research 19-04-00888 and the President of the Russian Federation for young scientists MK-420.2020.4.
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This work was carried out in accordance with state assignment AAAA-A19-119112290009–1 and supported by grants from the Russian Foundation for Basic Research 19–04-00888 and the President of the Russian Federation for young scientists MK-420.2020.4.
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ONO developed the concept and design of the study; GVS carried out experiments, analysed data, and wrote the manuscript; NGM and AVT carried out experiments and analysed the experimental results. All authors read and approved the final version of the manuscript.
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10482_2021_1693_MOESM1_ESM.pdf
Supplementary Fig. 1 Porin OmpF is involved in the entrance of ciprofloxacin (CF) into E. coli cells growing on glucose. In mid-log phase (OD600 of 0.4), bacteria E. coli growing in M9 minimal medium with glucose, succinate or acetate were treated with 3 µg CF ml−1 and the OD600 and CF concentration in the medium were monitored for 2 h. CF concentration was measured by its fluorescence (λex 271 nm and λem 416 nm) in samples taken through a membrane filter and determined using calibration curves at various OD600 values of the culture. The rate of ciprofloxacin absorption from the medium was expressed in µg/OD600 min. Values are the means and standard error (vertical bars) from at least three independent experiments. Statistical differences compared to the cells growing on glucose (P < 0.05) are noted with asterisk. (PDF 67 kb)
Supplementary Fig. 2
Sensitive pH recording makes it possible to track changes in substrate consumption caused by ciprofloxacin (CF) in wt (a) and recA (b) cells. Changes in ATP concentration under CF exposure in wt (c) and recA (d) cells. E. coli was grown in M9 minimal medium with glucose, succinate or acetate. Ciprofloxacin (0.3 or 3 μg ml−1) was added at OD600 of 0.4 at the time indicated by the arrow. Each experiment was repeated at least three times. The data shown are representative for pH measurements (a, b) or are presented as the means and standard error (vertical bars) for ATP determination (c, d). Statistical differences compared to the cells growing on glucose (P < 0.05) are noted with asterisk. (PDF 165 kb)
Supplementary Fig. 3
Production of extracellular superoxide (a) and H2O2 (b) and the effect of H2O2 addition on membrane potential (c), katG::lacZ expression (d), and survival (e) during cultivation of E. coli on glucose, succinate, and acetate. The rate of extracellular superoxide production (pmol /0.1 OD600 • min) and H2O2 accumulation were measured for 2.5 h. Values are the means and standard error (vertical bars) from at least three independent experiments. Statistical differences compared to the cells growing on glucose (P < 0.05) are noted with asterisk. (PDF 94 kb)
Supplementary Fig. 4
Deletion of the cysM gene encoding cysteine synthase B (CysM) did not affect ciprofloxacin-induced changes in the specific growth rate (a) and CFU (b) compared to the wild type strain. 3 µg CF ml−1 was added to E. coli cells growing in M9 minimal medium with glucose at OD600 of 0.4. The time of CF addition is indicated by an arrow. Values are the means and standard error (vertical bars) from at least three independent experiments. (PDF 77 kb)
Supplementary Fig. 5
DMSO slowed down the growth of wild type (a) and oxyR mutant (b) on LB medium. Catalase (500 U ml−1) or DMSO (5%) was added to LB medium prior to cell inoculation. The cultures were grown with shaking (150 rpm) at 37 °C for 3.5 h. Values are the means and standard error (vertical bars) from at least three independent experiments. (PDF 76 kb)
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Smirnova, G.V., Tyulenev, A.V., Muzyka, N.G. et al. Study of the contribution of active defense mechanisms to ciprofloxacin tolerance in Escherichia coli growing at different rates. Antonie van Leeuwenhoek 115, 233–251 (2022). https://doi.org/10.1007/s10482-021-01693-6
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DOI: https://doi.org/10.1007/s10482-021-01693-6