Changes in the growth rate and energy parameters of cells
Mutants lacking the rpoS, recA, ompF and tolC genes grew at the same rate, approximately 0.68 h− 1, as the parent when glucose was used as a substrate (Fig. 1a). Growing all strains on less energetically efficient succinate and acetate reduced the specific growth rate by 1.5 and 3.6 times, respectively. Interacting with DNA gyrase, ciprofloxacin inhibits replication and transcription, which leads to a decrease in the growth rate. The effect on growth rate was less pronounced at 0.3 µg CF ml− 1, where there was a 30-minute delay in the fall of µ, while when using 3 µg CF ml− 1, the growth rate began to decrease immediately after its addition in all strains studied, except ompF (Fig. 1b, c, d). For both concentrations of ciprofloxacin, the antibiotic effect on growth decreased with a decrease in the initial growth rate and was minimal in cultures grown on acetate. The absence of porin OmpF significantly slowed down the ciprofloxacin-induced decrease in µ during growth on glucose and succinate compared to the parent, which apparently reflects a decrease in the intensity of antibiotic entry into cells in this mutant (Fig. 1d). Indeed, measurements showed that the decrease in the concentration of ciprofloxacin in the medium of the parental strain growing on glucose was 2.4 times faster than in the ompF mutant (Fig. 1S). The recA mutation did not affect the growth rate changes induced by 0.3 µg CF ml− 1 (Fig. 1b). However, unlike the parent, recA deficiency prevented the specific growth rate from falling below zero after 90 minutes of exposure to 3 µg CF ml− 1 (Fig. 1c). The absence of the rpoS and tolC genes did not significantly affect the growth rate in the presence of both antibiotic concentrations.
In our previous work, we showed that ciprofloxacin induces phase alterations in E. coli respiration during growth in a minimal medium with glucose: the first phase strongly depends on antibiotic concentration; the second phase is SOS-dependent (Smirnova et al. 2017). In the current study, dissolved oxygen (dO2) was continuously measured directly in cultures grown with glucose, succinate and acetate. Despite the constant rotation of the flasks, the concentration of dissolved oxygen in untreated cultures gradually decreased due to an increase in oxygen consumption with an increase in biomass (control curves in Fig. 2a, b). The rate of this decrease, expressed as dO2/OD600 ∙ min, was 1.12 ± 0.04, 0.66 ± 0.01 and 0.16 ± 0.01 for glucose, succinate and acetate, respectively. The lower basal level of dO2 in cultures growing with succinate (Fig. 2a, b) is explained by a sharp decrease in dO2 by 30% within 10 minutes after the inoculum was added to the medium with succinate. The effect of ciprofloxacin on dO2 depended on both the type of growth substrate and the concentration of the antibiotic and correlated with its effect on the growth rate. After treating wild-type cells with 0.3 µg CF ml− 1, dO2 continued to decline over approximately 40, 60 and 90 minutes in E. coli grown with glucose, succinate and acetate, respectively (Fig. 2a). In the case of acetate, even a slight acceleration of oxygen consumption was observed in comparison with the control. After 80 min exposure to 0.3 µg CF ml− 1, an increase in dO2 was observed in cultures grown with glucose and succinate (Fig. 2a). This phase was SOS-dependent and did not occur in the recA mutant (Fig. 2b). Cultures grown on succinate and acetate did not show a phase of a sharp increase in dO2 immediately after treatment with 3 µg CF ml− 1, which is a characteristic feature of a culture grown on glucose and corresponds to a sharp inhibition of growth. The SOS-dependent phase of respiration inhibition by 3 µg CF ml− 1 in cultures grown on succinate and acetate was more gradual than in cultures grown on glucose (Fig. 2a, b).
Metabolic shifts caused by ciprofloxacin can also be monitored using continuous, sensitive pH recording. The consumption of the substrate was accompanied by acidification of the medium due to the accumulation of acid by-products during growth on glucose, while alkalisation of the medium occurred during growth on succinate and acetate (Fig. 2S). Ciprofloxacin dose-dependently suppressed glucose consumption, but the consumption of acetate and succinate continued at a rate that did not differ significantly from the control. Interestingly, despite the same growth rate as the wild type, the recA mutant showed lower substrate consumption (Fig. 2S-b) as well as a slower decrease in dO2 during growth on all studied substrates (Fig. 2b).
To determine the ability of cells to maintain membrane potential, we used the fluorescent dye DiBAC4 (3), which stains only depolarized cells (Wickens et al. 2000). The percentage of depolarized cells in untreated cultures was higher during growth on succinate and acetate, 2.7 ± 0.1 and 2.2 ± 0.1, respectively, compared to 0.9 ± 0.1 during growth on glucose. Cultures exposed to 3 µg CF ml− 1 maintained the level of depolarized cells below 10% for an hour, but then there was a rapid decrease in membrane potential (Fig. 2c). In accordance with our previous work (Smirnova et al. 2017), this phase was SOS-dependent and coincided with the SOS-dependent phase of respiratory inhibition. The percentage of depolarized cells was 5 times lower in the recA mutant compared to the parental strain, when both grow on glucose (Fig. 2c). Wild-type cells growing on glucose lost Δψ 2 times faster than cells growing on succinate and acetate (Fig. 2c), indicating more severe damage under conditions that provide a higher growth rate. Treatment with chloramphenicol (25 µg ml− 1) or a bacteriostatic dose of H2O2 (2 mM) caused small changes (below 4%) in Δψ, while the use of a bactericidal dose of H2O2 (10 mM) led to the appearance of a phase of rapid decrease in the membrane potential (Fig. 2d). These data show that bacteriostatic drugs do not induce the late phase of the SOS response, in which the loss of membrane potential is one of the main features (Erental et al. 2014).
The growth rate of bacteria is largely determined by the rate of production of NADH, which is a source of reducing equivalents in the respiratory chain for creating an electrochemical proton gradient and ATP synthesis with the participation of ATP synthase. The steady-state level of NADH, which results from its production and consumption, was even lower and, accordingly, the NAD+/NADH ratio was higher in cells growing on glucose than in cells growing on succinate and acetate (Fig. 3a). 20 min after the addition of 3 µg CF ml− 1, there was a decrease in NADH and a 2-fold increase in the NAD+/NADH ratio in comparison with the untreated culture on glucose, while the level of NADH on succinate increased 1.7 times (Fig. 3a, b). No changes in these parameters were observed on acetate. Intracellular ATP increased after ciprofloxacin treatment in a dose-dependent manner (Fig. 3c). For all substrates, this increase was gradual at 0.3 µg CF ml− 1 and, conversely, quickly peaked at 3 µg ml− 1 (Fig. 3d), which coincided with the growth inhibition mode at various concentrations of ciprofloxacin (Fig. 1b, c). The recA mutant showed the same mode of ATP changes as the parental strain (Fig. 3d).
Changes in the redox state of cells growing with various sources of carbon and energy
In our previous work, we showed that after a 20-min exposure, ciprofloxacin reduced both the production of extracellular superoxide and the level of extracellular H2O2, but significantly increased the level of intracellular (GSHin) and extracellular glutathione (GSHout) in cells growing on glucose (Smirnova et al. 2017). Growing on succinate had no effect on ROS generation compared to glucose, while the use of acetate reduced superoxide production by 1.5 times and the concentration of H2O2 in the medium by 2.2 times (Fig. 3S). The basal level of GSHin was 1.3 times lower for succinate and 1.4 times higher for acetate as compared to cells grown on glucose (Fig. 4a). With all substrates, the intracellular glutathione level gradually increased during the observation period. In untreated cells, about 10% of the total synthesized glutathione was excreted into the medium, where its concentration per biomass unit was maintained at an approximately constant level (Fig. 4b).
Treatment with 3 µg CF ml− 1 caused a transient increase in GSHin level that was proportional to the degree of growth inhibition and had significantly lower amplitude in cells growing on acetate and succinate compared to glucose (Fig. 4a). The increase in the level of GSHin was accompanied by an increase in its extracellular concentration, which was higher for glucose and succinate than for acetate (Fig. 4b). The release of glutathione was especially accelerated after 60 min of exposure to ciprofloxacin. This phase coincided with a drop in the intracellular GSH and phases of SOS-dependent inhibition of respiration and rapid decrease in membrane potential (Fig. 2a, c). To clarify the role of the SOS-response in altering glutathione levels, we measured intracellular and extracellular glutathione in the recA mutant. Unexpectedly, an inversion of GSH levels inside and outside the cells was observed: GSHin was low and stable during observation, while GSHout was 4.5 times higher and gradually increased with increasing biomass (Fig. 4c). That is, most of the glutathione in this mutant left the cells as it was synthesised. The addition of 3 µg CF ml− 1 did not change the level of GSHin, but accelerated its release into the medium.
Thus, ciprofloxacin provokes an acceleration of GSH synthesis in wild-type and mutant cells, which is apparently associated with a sharp inhibition of protein synthesis and transient excess of cysteine, which is a limiting component for GSH synthesis in a minimal medium. We have previously shown that this behavior of glutathione is characteristic of the inhibition of protein synthesis by valine and chloramphenicol, where GSH serves as a buffer for the bulk of excess cysteine (Smirnova et al. 2019). Simultaneously with an increase in glutathione production, a sharp inhibition of protein synthesis leads to an abrupt release of sulfide into the medium. Only a large dose of ciprofloxacin (3 µg ml− 1) caused the release of H2S in E. coli growing on glucose (Tyulenev et al. 2018). However, this dose of ciprofloxacin did not induce H2S release when cells were grown on succinate or acetate (Fig. 4d). As in the case of valine and chloramphenicol (Smirnova et al. 2019), H2S generation under exposure of E. coli to ciprofloxacin depended on the activity of cysteine synthase B (CysM) and was absent in the cysM mutant (Fig. 4e). H2S production was also absent in the recA mutant (Fig. 4e). It was previously reported that H2S can protect cells against antibiotic killing (Shatalin et al. 2011). To clarify this possibility under our conditions, we studied the effect of ciprofloxacin on the growth rate and the number of CFU in the cysM mutant with abolished H2S production. No significant differences were found between the wild-type strain and the cysM mutant (Fig. 4S).
Expression of antioxidant genes in E. coli growing with various carbon and energy sources
Several groups have shown a correlation between quinolone lethality and the accumulation of ROS (reviewed by Bush et al. 2020). When ROS accumulate in the cell, bacteria respond through the OxyR, SoxRS and RpoS regulons, which control the transcription of genes encoding ROS-scavenging enzymes (Imlay 2013). The expression of genes katG and katE, encoding catalases HPI (KatG) and HPII (KatE), is also positively regulated by RpoS (Ivanova et al. 1994), the level of which is inversely proportional to the specific growth rate (Ihssen, Egli 2004). In accordance with these data, in our experiments, the expression of katG::lacZ increased 1.4 and 2.3 times with growth on succinate and acetate, respectively, compared with glucose (Fig. 5a). Under the same conditions, the expression of katE::lacZ increased 1.8 and 2.1 times (Fig. 5b). The dependence of the expression of both genes on the type of substrate and thus on the growth rate was sharply reduced in the rpoS mutant (Fig. 5a, b). The expression of the sulA gene, which is controlled by SOS-regulon, also increased with a decrease in the growth rate of the culture in the sequence: glucose, succinate, acetate (Fig. 5c). This growth rate dependence of sulA expression may be caused by an increase in the level of (p)ppGpp in slower growing cells, which leads to the induction of genes involved in the SOS response, as shown for the E. coli stringent response (Durfee et al. 2008). The rpoS mutation did not significantly affect the expression of sulA::lacZ during growth on glucose, but additionally increased it by 1.7 and 2.4 times on succinate and acetate, respectively. RpoS is known to positively regulate DNA polymerases II (polB) and IV (DinB) independently of LexA (Maslowska et al. 2019); therefore, additional induction of the SOS response in the rpoS mutant may be a compensatory mechanism for maintaining their required level.
We have previously shown that the expression of the antioxidant genes sodA and katG, as well as the level of total catalase activity, significantly decreased during the action of ciprofloxacin on E. coli growing in a minimal medium with glucose (Smirnova et al. 2017). Ciprofloxacin treatment of cells growing on succinate or acetate also resulted in a decrease in katG::lacZ expression (Fig. 5d), while an increase in its expression in response to H2O2 addition was observed on all substrates (Fig. 5S). During growth on acetate, the expression of the sodA gene was 1.2 times higher than on glucose and succinate, and was maintained approximately constant in untreated culture (Fig. 5e). The higher basal level of sodA expression during growth on acetate, apparently, is a consequence of the removal of the repression of this gene by the ArcAB regulatory system under conditions of an increased level of dO2 (Compan and Touati 1993). Treatment of cells with 3 µg CF ml− 1 reduced the expression of sodA::lacZ during growth on all tested substrates. The effect of ciprofloxacin on the expression of the katE gene was proportional to the degree of growth inhibition by the antibiotic: it increased with an increase in the concentration of ciprofloxacin and with the transition to a more energy-efficient substrate. The addition of 3 µg CF ml− 1 increased katE expression 1.5-fold when cells grew on glucose, 1.2-fold if growth was on succinate and had no significant effect during growth on acetate (Fig. 6S). Thus, regardless of the substrate used, we did not observe an increase in the expression of antioxidant genes katG and sodA when cells were treated with ciprofloxacin. The increase in katE::lacZ expression under these conditions was the result of the inhibitory effect of the antibiotic on the growth rate of bacteria.
Consistent with previous data, ciprofloxacin treatment caused an immediate activation of the SOS response (Theodore et al. 2013). Regardless of the substrate used, the increase in sulA::lacZ expression was more pronounced with a lower antibiotic dose (Fig. 5f). Perhaps the lower level of sulA induction with 3 µg CF ml− 1 (3 times) compared to 0.3 µg CF ml− 1 (10 times) was associated with a sharper inhibition of transcription and translation at a high concentration of the antibiotic.
Effect of ompF, tolC, rpoS, and recA mutations on ciprofloxacin lethality
Among all tested mutations, only mutations in the recA and ompF genes could significantly affect the lethal activity of ciprofloxacin, as follows from the analysis of killing curves (Fig. 6). The effect of ompF deficiency strongly depended on the concentration of ciprofloxacin. At 0.3 µg CF ml− 1, its bactericidal activity decreased by 2 times in cells growing on glucose or succinate (Fig. 6a, b), while at 3 µg CF ml− 1, it increased 9, 3, and 2 times during growth on glucose, succinate, and acetate, respectively, compared to the parental strain (Fig. 6d, e, f). RecA deficiency reduced the number of CFUs by 2–4 orders of magnitude compared to the parental strain, depending on the concentration of ciprofloxacin and the substrate used. The absence of RpoS only slightly (1.7 times) increased the lethal activity of ciprofloxacin when cells were grown on succinate at both antibiotic concentrations or on glucose at 0.3 µg CF ml− 1 (Fig. 6a, b, e). The tolC mutant showed the same tolerance to ciprofloxacin as the parent.
The bactericidal activity of quinolones, including ciprofloxacin, is biphasic: the lethality of the drugs increases to a concentration known as the optimal bactericidal concentration (OBC), after which the bactericidal activity then declines (Lewin et al. 1991). In wild type E. coli, the OBC is about 0.3 µg CF ml− 1. Accordingly, 0.3 µg CF ml− 1 showed a higher bactericidal activity (up to one order of magnitude) than 3 µg CF ml− 1 during the growth of wild-type cells and rpoS and tolC mutants on all tested substrates. In contrast, the dose 3 µg CF ml− 1 was more lethal for the recA mutant, regardless of the substrate used. Significant changes in the bactericidal activity of various concentrations of ciprofloxacin were also observed for the ompF mutant. Paradoxically, ompF deficiency increased tolerance to 0.3 µg CF ml− 1, but enhanced sensitivity to 3 µg CF ml− 1. This may be due to a shift in the OBC for this mutant towards a higher dose of ciprofloxacin compared to the parental strain due to a decrease in the entry of the antibiotic into the cells. In the ompF mutant, the dose of 3 µg CF ml− 1 corresponded to the OBC, causing a maximum decrease in CFU; a further increase in the concentration to 10 µg CF ml− 1 was accompanied by a decrease in the lethality of the antibiotic (not shown).
Depending on the substrate used, the bactericidal activity of the antibiotic decreased in the sequence: glucose, succinate, acetate, regardless of the E. coli strain and the concentration of ciprofloxacin (Fig. 6). Earlier, we showed for a wild-type strain that there is an inverse correlation between logCFU ml− 1 and the specific growth rate of bacteria growing on different energy sources (Smirnova and Oktyabrsky 2018). Our current work has revealed the existence of such a relationship for all studied mutants (Fig. 7). The degree of shift of the graphical dependence of logCFU ml− 1 on the specific growth rate along the ordinate in the mutant relative to the wild-type strain reflects the contribution of the corresponding gene to the tolerance to ciprofloxacin. All strains showed the highest tolerance when growing on acetate, which provides the lowest growth rate. The protein synthesis inhibitor chloramphenicol reduced the growth rate to 0.1 h − 1 within 60 min after its addition and caused an increase in ciprofloxacin tolerance similar to that of cells growing on acetate (Fig. 7S). These data confirm that a low growth rate leads to high tolerance, regardless of the reasons that lead to slower growth.
Oxidative stress is thought to be involved in the lethal effects of ciprofloxacin (Hong et al. 2020). However, the absence of RpoS, which controls the induction of H2O2 scavengers KatG and KatE and the ferrous sequestering protein Dps (Navarro Llorens et al. 2010), only slightly affected the bactericidal activity of this antibiotic (Fig. 6). To test the role of RpoS in protection against oxidative stress under our conditions, we studied the bactericidal activity of H2O2 in E. coli cells growing at different growth rates. Treatment of cells growing on glucose with 10 mM H2O2 completely suppressed the growth of both the parental strain and the rpoS mutant, while when cells were cultured on acetate or succinate, gradual growth recovery was observed (Fig. 8Sa, b). Accordingly, this dose of H2O2 did not kill bacteria growing on succinate and acetate, but showed significant lethality when growing on glucose (Fig. 8Sc). Under these conditions, the rpoS mutant was 3 orders of magnitude more sensitive to H2O2 than the parent. Prevention of the lethal effect of H2O2 in slowly growing cells was not the result of induction of the RpoS regulon, as this was observed in both the parental strain and the rpoS mutant.
We also tested the involvement of oxidative stress in killing E. coli during stress recovery after plating ciprofloxacin-treated cells on LB agar without antibiotic. The presence of catalase in LB agar did not affect the CFU ml− 1 in the wild-type strain treated with 0.3 µg CF ml− 1 (Fig. 8). However, in line with previous data (Hong et al. 2020), the addition of DMSO to LB agar increased logCFU ml− 1 by an order of magnitude. It is known that DMSO traps hydroxyl radicals; therefore, it is possible that oxidative stress is involved in the killing of this part of cells. The procedure for determining the colony forming ability (washing, serial dilutions, and inoculation on the agar surface) involves a change in the composition of the medium, temperature and oxygen concentration and itself represents a series of stresses for the cells. Apparently, defense mechanisms in wild-type cells make it possible to avoid the damaging effect of these stresses. In particular, the addition of catalase and DMSO to agar has no effect on the change in logCFU ml− 1 in the control culture (Fig. 8a). In contrast, the plating efficiency of the oxyR mutant was increased by two orders of magnitude with the addition of catalase and by one order of magnitude when the LB agar contained DMSO (Fig. 8b, control curves). These data indicate that the plating bacteria itself induces oxidative stress if the antioxidant system is compromised. In the oxyR mutant, the CFU ml− 1 decreased by 5 times when cells after treatment with ciprofloxacin were plated on LB agar without additives, and by 10 and 100 times when catalase or DMSO was added (Fig. 8b). That is, the killing activity of ciprofloxacin even increased in parallel with an increase in plating efficiency of the oxyR mutant in the presence of catalase and DMSO. It is likely that oxidative stress can lead to additional DNA damage during plating of cells on agar without antibiotic, which complicates the repair of double-stranded DNA breaks formed when the DNA gyrase-ciprofloxacin complex is detached from the DNA. At the same time, in contrast to catalase, the presence of DMSO in the LB medium significantly slowed down the growth of bacteria, especially in the first two hours of cultivation (Fig. 9S). Therefore, it is possible that the protective effect of DMSO in the wild-type strain is associated not only with the direct uptake of hydroxyl radicals, but with an increase in the efficiency of successful repair of DNA damaged by ciprofloxacin in slower growing cells.