According to traditional concepts, low-molecular-weight thiols, hydrogen sulfide, cysteine, and glutathione protect wild-type bacterial cells from oxidative stress [1, 3, 23]. However, against the background of inactivation of ADP-heptose synthesis in E. coli with a deletion of gmhA oxidative stress develops, accompanied by a decrease in the pool of reducing NADPH equivalents and the reductive capacity of the cytoplasm [7]. Such a redox imbalance, apparently, leads to an increase in the amount of oxidized forms of low-molecular-weight thiols in cells and, as a result, to a depression (decrease in efficiency) of protective mechanisms involving thiols. In addition, a sharp decrease in the intracellular pool of cysteine and generation of H2S, accompanied by an increase in the level of reduced thiols, in the cells of the ∆gmhA mutant [7] may indicate a redistribution of sulfur-containing metabolites and possible redox modifications of proteins.

In this work, we found that the restriction of the metabolism of cysteine (∆cysB and ∆cysE) and inhibition of glutathione synthesis (∆gshAB) did not lead to the expected increase, but to a decrease in the sensitivity of the ∆gmhA mutant to antibiotics, which is especially pronounced in the case of such a bactericidal antibiotic as nalidixic acid. The results we obtained suggest that the inactivation of ADP-heptose biosynthesis in E. coli provokes the development of oxidative stress, which is accompanied by cascade irreversible redox modifications of proteins with the participation of cysteine and glutathione. A decrease in the total concentration of low-molecular-weight thiols limits the processes of glutathionylation and cysteinylation of various enzymes, removing the toxic effect and increasing the resistance of the ∆gmhA strain to the action of antibacterial drugs. Redox-sensitive cysteine residues are often included in the catalytic centers of enzymes of central metabolism, in particular, enzymes of the pentose phosphate pathway, transketolase and ribulose-5-phosphate epimerase [24, 25]. A decrease in the catalytic activity of such enzymes or their complete inactivation can dramatically affect the ability of cells to carry out vital anabolic processes and reduce the adaptive potential of cells. In addition, since the level of cysteine must be maintained within 0.1–0.2 mM to prevent genotoxicity, and its pumping out of the cell requires energy consumption [4], a decrease in the cysteine pool can save cell resources (ATP and NADPH), which is critical when they are deficient, as observable, as we showed earlier [7], in mutants with a gmhA deletion. In fact, we observed a decrease in the proportion of oxidized glutathione in ∆gmhA ∆cysE, ∆gmhA ∆cysB, and ∆gmhA gshAB E. coli strains, which indicates the effective recovery of available glutathione in these strains, despite the decrease in the total amount of glutathione (Fig. 3).

Blocking the export of cysteine (∆eamA) or an increase in imports (Ptet-tcyP) into the cells of cystine, which is an oxidized form of cysteine, leads to an even greater increase in the sensitivity of cells with a gmhA deletion to antibiotics. This is due to additional depletion of the NADPH pool, which is already significantly reduced when ADP-heptose biosynthesis is inhibited [7], since the accumulation of cystine in the cell should increase the consumption of reducing equivalents (NADPH) for the reduction of cystine to cysteine [22]. An increase in the concentration of cystine is dangerous for the cell, since cystine promotes the formation of disulfide bridges in proteins, which affects their functioning [27]. For example, cystine activates alkaline phosphatase in this way [27]. Therefore, the cell tries to quickly restore cystine, which, as a result, is present in small quantities under normal conditions [28]. As a result, inhibition of the cystine importer by the product (cystine) does not occur and it continues to be pumped into cells. The observed increase in the proportion of the oxidized form of glutathione GSSG with ∆eamA (Fig. 3b) is due to an increase in the content of cystine in the cell, which contributes to an increase in the oxidation of thiol groups with the formation of disulfide bridges. Exceeding the normal intracellular concentration of cysteine due to its recovery from cystine or blocking export is also dangerous for the cell, as it causes an increase in ROS, and also activates the Fenton genotoxic reaction [2, 4]. In addition, at high concentrations, cysteine can compete with threonine for the active site of threonine deaminase, the first enzyme in the isoleucine biosynthesis pathway [27]. It should be noted that an increase in the content of cystine due to oxidative stress also leads to an increase in the oxidized form of glutathione GSSG. It is known that the Lrp transcription factor reacts to excess cysteine and induces AlaE, a multifunctional transporter that pumps cysteine out of the cell until its level can be reduced. The reduction of cystine and the removal of cysteine from the cell significantly increase the need for NADPH in cells [27]. Thus, our induced increase in cystine entry increases NADPH consumption [27], while slowing the release of cysteine leads to the reduction of iron and genotoxicity [4]. In both cases, the growth of the oxidized form of glutathione (GSSG) is observed (Fig. 3), which can directly interact with the thiol groups of proteins, causing their glutathionylation [29]. In addition, a decrease in the level of NADPH leads to the activation of protein glutathionylation by glutaredoxin [24, 29]. It should also be noted that in order to cope with the excess of cystine and cysteine, the cell needs to consume ATP, whose content in the mutants with the gmhA deletion is reduced by 2 times [7]. Another mechanism for increasing sensitivity to antibiotics may be a violation of the oxidative folding of proteins in the periplasm. E. coli. Thus, we believe that the main reason for the increase in sensitivity to antibiotics of the strain with the gmhA deletion with an increase in the level of cystine and cysteine, there is a depletion of the cell’s energy resources and redox modification of proteins.

The introduction of the studied mutations in all cases leads to a decrease in the total intracellular pool of thiols, which is increased due to inactivation of the synthesis of ADP-heptose in cells with the gmhA deletion. This is a predictable result due to a significant decrease in cysteine levels and glutathione in cells, and disruption of cystine transport creates an excess of oxidized forms that require NADPH, which reduces the cell’s ability to restore thiol groups. The greatest effect is achieved in the case of the cysE deletion. In addition, this deletion completely cancels the increase in ROS production caused by the gmhA deletion, while all other mutations in the genes that regulate the metabolism of cysteine and glutathione, as well as a violation of cystine transport, lead to an even greater increase in the level of ROS in cells with a gmhA deletion. Because O-acetylserine, the reaction product catalyzed by acetylserine transferase, activates the CysB regulatory protein and is also an intermediate in the cysteine biosynthesis pathway, then a quite understandable more significant suppression of the gmhA deletion effect occurs in cells with ΔcysE compared with ΔcysB.

Thus, no correlation was found between the cytotoxic effect of antibiotics and the level of ROS, the total pool of thiols, or the viability of the initial cell population. However, a correlation was found between an increase in antibiotic sensitivity and an increase in the proportion of oxidized glutathione (GSSG). In mutants with greater sensitivity to antibiotics, the level of GSSG is higher, and in mutants with reduced sensitivity it is lower. The data we obtained indicate that the reason for the decrease in the sensitivity of cells with a gmhA deletion to antibiotics is the depletion of the pool of low-molecular-weight thiols, as is observed in violation of the metabolism of cysteine and glutathione, while an increase in the content of cystine, the oxidized form of cysteine, or cysteine leads to an increase in the sensitivity of these cells to antibiotics.