Interplay between mistranslation and oxidative stress in Escherichia coli
| 29. Juni 2024
Article Category: Original article
Online veröffentlicht: 29. Juni 2024
Seitenbereich: 147 - 154
Eingereicht: 01. März 2024
Akzeptiert: 01. Mai 2024
DOI: https://doi.org/10.2478/aiht-2024-75-3834
Schlüsselwörter
© 2024 Valentina Ević et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Translation is a fundamental cellular process in which the nucleotide sequence of mRNA is translated into the amino acid sequence of a protein. The important role in this process is played by aminoacyl-tRNA synthetases (aaRSs), enzymes which covalently link amino acids to cognate tRNAs. The resulting aminoacyl-tRNAs (aa-tRNA) serve as substrates during translation on the ribosome (1, 2). Additionally, many aaRSs are multifunctional proteins participating in various cellular processes beyond translation, including stress response (3, 4, 5, 6, 7, 8).
Aminoacylation is a two-step reaction. The first, activation step, involves adenosine triphosphate (ATP) to form an aminoacyladenylate (aa-AMP) intermediate. The second step involves the transfer of the aminoacyl moiety to cognate tRNA. Due to the structural and chemical similarity of some amino acids, certain aaRSs can mistake non-cognate for cognate amino acids, which can lead to their incorporation in the protein and result in mistranslation (mistake in translation). To reduce the rate of mistranslation, aaRSs correct their mistakes through editing reactions, which can occur before or after the transfer of the aminoacyl group to the tRNA (9, 10). Pre-transfer editing involves the hydrolysis of non-cognate aa-AMP in the synthetic site (where aminoacylation takes place), while post-transfer editing involves the hydrolysis of non-cognate aa-tRNA in a specialised editing domain.
Isoleucyl-tRNA synthetase (IleRS) charges tRNAIle with cognate amino acid isoleucine (Ile) enabling its incorporation in the proteins. IleRS has been known to mistake proteinogenic amino acid valine (Val) and non-proteinogenic amino acid norvaline (Nva) for Ile (11, 12). Nva is a non-canonical amino acid and a side-product of leucine biosynthesis in bacteria (13). To repair the result of non-cognate amino acid misrecognition IleRS uses both pre-transfer editing of non-cognate aa-AMP within the synthetic site and deacylation of misaminoacylated tRNAIle in the post-transfer editing domain (11, 12, 14, 15).
Mistranslation is usually toxic as it leads to the production of functionally aberrant and misfolded proteins, which is often associated with adverse effects due to protein aggregation in the cell, impaired cell fitness and growth, morphological changes, and even cell death (16, 17, 18). In yeast, mistranslation disrupts the mitochondrial function (19), in a mouse model misfolded proteins cause neurodegenerative diseases (20), while mutations increasing the rate of mistranslation are linked to neuropathies in humans (21, 22). Aminoglycoside antibiotics, such as streptomycin and kanamycin, kill bacteria because they induce mistranslation and protein misfolding (23). In
However, there are several examples in which mistranslation has a beneficial effect (25, 26). Naturally occurring mistranslation of specific amino acids can be induced as part of response to stress. A most notable example is mismethionylation induced under oxidative stress, which is found in all three domains of life (25, 27). It stems from the ability of methionine to act as a sink for reactive oxygen species (ROS), since it can be reversibly oxidised to methionine sulphoxide (28). Constitutively higher mistranslation rates are common in parasites, such as
Reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radicals, are by-products of aerobic metabolism and play an important role in cell signalling in many organisms, including bacteria (32, 33). However, if the metabolism is disrupted, ROS levels increase beyond the cell's capacity to clear them, which causes oxidative stress (34). Additionally, oxidative stress can be caused by other bacteria that produce and excrete ROS or by plant cells and animal immune cells that produce ROS in defence against pathogenic bacteria (35). Furthermore, oxidative stress may be a result of microbe exposure to heat, metals, solvents, and clinical antibiotics (35). Whichever the cause, oxidative stress damages nucleic acids, lipids, and proteins and, ultimately, kills the cell (34). It also has diverse effects on the aaRS function. In most cases it impairs the aaRS activity (36, 37, 38, 39), but some aaRSs are resistant to oxidative conditions (40, 41, 42, 43).
We know little about how mistranslation affects response to oxidative stress in the
The wild-type
Both strains were grown at 37 °C in the M9 minimal medium (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 2 mmol/L MgSO2, 0.1 mmol/L CaCl2, 0.4 % glucose, 50 μmol/L thiamine, 100 μmol/L Ile, 100 μmol/L Leu, and 100 μmol/L Val (hereinafter: enriched M9 medium). Considering that higher concentrations of Val inhibit isoleucine biosynthesis, which causes isoleucine pseudo-auxotrophy in strains derived from
To compare the growth of the wild-type and the mutant strain with the editing-deficient IleRS in mistranslating conditions, i.e. in the presence of Val or Nva, overnight cultures grown in the enriched M9 medium were diluted to OD600=0.04 and grown for another 10 h in the enriched M9 medium either supplemented with 0.75 or 1.5 mmol/L Val or Nva or without the amino acid supplements.
To compare the growth of each strain under oxidative stress, overnight cultures were diluted to OD600=0.04 in the enriched M9 medium supplemented with various concentrations of H2O2 (0, 0.25, 0.5, 0.75, or 1 mmol/L) and grown further for 10 h.
To compare the effects of both mistranslation and oxidative stress overnight cultures of both strains were diluted to OD600=0.04 in the enriched M9 medium either supplemented with 0.75 or 1.5 mmol/L Val or Nva or without the amino acid supplements. Diluted cultures were divided in two tubes and further cultured for 10 h. Culture in one tube continued to grow in the same medium, while H2O2 was added to a final concentration of 0.5 mmol/L to the other tube to induce oxidative stress.
To compare the effect of pre-incubation with Val and Nva before the addition of H2O2, overnight cultures of both strains were diluted to OD600=0.04 with or without Val or Nva (0.75 mmol/L or 1.5 mmol/L) to induce mistranslation and were incubated to reach the early exponential phase (OD600=0.2) or mid-exponential phase of growth (OD600=0.5). At that point, each culture was divided in two samples and 0.5 mmol/L of H2O2 was added to one to induce oxidative stress. The growth of bacteria was further monitored for up to 10 h since the initial dilution of overnight cultures.
In all experiments growth curves were determined by measuring OD600 over 10 h with the Ultrospec 10 cell density meter (Amersham Biosciences, Piscataway, NJ, USA). Due to the limitations of the instrument, OD600 values above 2.0 could not be measured. All experiments were repeated at least two times.
Figure 1 shows the 10-hour growth curves of both the wild-type and editing-deficient mutant IleRS(Ala10) ED─ in the enriched M9 medium supplemented with 0.75 or 1.5 mmol/L Val or Nva or not (controls). Val and Nva had a small inhibitory effect on the growth of the wild-type strain, while the inhibition was more pronounced in the editing-deficient strain incubated with both Nva concentrations or 1.5 mmol/L Val. This supports an earlier report that the absence of IleRS editing activity affects the growth due to elevated mistranslation rates when exogenous non-cognate amino acids are present (12). Interestingly, in the presence of 0.75 mmol/L Val the IleRS(Ala10) ED─ strain growth curve was very similar to the growth curves of the wild-type strain in the presence of both Val concentrations.
Figure 1
Representative growth curves of at least two biological replicates of the wild-type
Figure 2 shows that both the wild-type and the mutant strain were sensitive to H2O2, which inhibited the bacterial growth by prolonging the lag phase. During the lag phase, bacteria activate stress responses to defend themselves, after which they continue to grow (45, 46, 47, 48). At all H2O2 concentrations, the mutant exhibited a longer lag phase than the wild-type, which indicates that it took longer to deal with the oxidative stress.
Figure 2
Representative growth curves of at least two biological replicates of the wild-type
To determine the effects of elevated mistranslation rates induced by Val and Nva on the growth of the editing-deficient mutant strain under oxidative stress, we added 0.5 mmol/L H2O2 to the cultures, as this concentration had inhibited the growth of both strains but not as much as 0.75 and 1 mmol/L H2O2. Cell growth was monitored for 10 h (Figure 3). Unsupplemented with Val, Nva, or H2O2, both strains grew similarly, but supplementation with 0.5 mmol/L H2O2 prolonged the lag phase of both strains, albeit more evidently of the editing-deficient strain (Figure 3, top left panel).
Figure 3
Representative growth curves of at least two biological replicates of the wild-type
In the presence of 0.75 mmol/L Val and H2O2, the wild-type strain exhibited a longer lag phase compared to conditions without oxidative stress (Figure 3, top middle panel). Interestingly, however, after 10 h, the OD600 restored to the one measured in the wild-type culture supplemented with 0.75 mmol/L Val but not H2O2, which suggests successful recovery of the wild-type strain from oxidative stress. The editing-deficient mutant, in turn, showed slower growth in the presence of 0.75 mmol/L Val and H2O2, indicating that its growth is adversely affected by mistranslation in the oxidising conditions and does not recover like the wild-type strain.
Experiments with 0.75 mmol/L Nva yielded similar results (Figure 3, top right panel). Supplementation with higher Val and Nva concentrations did not improve the growth of the editing-deficient strain under oxidative stress compared to the culture unexposed to H2O2 or the wild-type culture under oxidative stress (Figure 3, bottom panels). It appears that the editing-deficient strain is more sensitive to H2O2 than the wild-type strain and that supplementation with Val or Nva and H2O2 additionally inhibits its growth.
Figure 4 shows that H2O2 added at the early exponential phase (OD600=0.2) delayed the growth of both strains when they were not pre-incubated with Val or Nva. However, the lag phase of both strains was shorter (Figure 4, top left panel) than when H2O2 was added immediately to the culture at OD600=0.04 (Figure 3, top left panel), indicating that the bacteria become more resilient to oxidative stress as the culture grows. When 0.75 mmol/L Val was present in the medium, H2O2 addition at the early exponential phase prolonged the lag phase of both strains (Figure 4, top middle panel). Comparison of OD600 at hour 10 showed that the editing-deficient strain slightly outgrew the wild type in oxidative conditions (Figure 4, top middle panel), indicating that pre-incubation with 0.75 mmol/L Val had a certain beneficial effect on the editing-deficient strain under oxidative stress, unlike in the experiment when Val and H2O2 were added simultaneously (Figure 3, top middle panel). Pre-incubation with Nva or 1.5 mmol/L Val, however, did not achieve a similar beneficial effect (Figure 4, top right, bottom left, and bottom right panels). One of the reasons may be that the cellular response to mistranslation stress did not have enough time to adapt cells to oxidative stress.
Figure 4
Representative growth curves of at least two biological replicates of the wild-type
Therefore, we conducted another experiment in which H2O2 was added when bacterial cultures reached the mid-exponential phase of growth (OD600=0.5). The resulting inhibition was small in both strains (Figure 5, top left panel). In the wild-type strain pre-incubated with 0.75 mmol/L Val the effect of H2O2 was minimal (Figure 5, top middle panel). As in the previous experiment, the editing-deficient strain eventually outgrew the wild-type under oxidative conditions (Figure 5, top middle panel) at hour 10. Interestingly, pre-incubation with 0.75 mmol/L Nva resulted in similar OD600 at hour 10 as the wild-type's (Figure 5, top right panel), which suggests that longer pre-incubation with non-cognate amino acids before the addition of H2O2 may improve response to oxidative stress through an adaptive effect of mistranslation. However, higher Val or Nva concentration (1.5 mmol/L) did not result in such an effective response (Figure 5, bottom left and right panels), which points to a delicate interplay between mistranslation and oxidative stress and even more delicate balance between deleterious and beneficial effects of mistranslation.
Figure 5
Representative growth curves of at least two biological replicates of the wild-type
To the best of our knowledge, there are no reports on organisms with editing-deficient aaRSs and their response to oxidative stress. Understanding the impact of mistranslation on the bacterial cell and its response to oxidative stress is important in biomedicine, because the immune system has a toxic effect on pathogenic bacteria via ROS (35). Therefore it is essential to elucidate the mechanisms of adaptation of bacteria to oxidative stress.
In general, mistranslation has toxic effect on cells and organisms (16, 17, 18), but some translational errors can be beneficial for a cell or organism under certain stress conditions (26). For example, global mistranslation of any amino acid caused by ribosomal errors leads to an increased expression of the sigma factor RpoS, a subunit of bacterial RNA-polymerase that directs RNA-polymerase to the promoters of genes associated with stress response (49). Among these genes are the genes coding for catalase KatE and peroxiredoxin OsmC, which are important for the recovery of cells exposed to hydrogen peroxide.
In this work we examined the effects of specific mistranslation at isoleucine codons and replacement of isoleucine with structurally similar Val or Nva on bacterial growth in oxidative conditions. To this end we used the
Ours is the first study of how mistranslation caused by IleRS errors affects bacterial growth under oxidative stress. The results show that simultaneous exposure of bacterial cells to two types of stresses (mistranslation and oxidative stress) is toxic and slows down their growth. However, if the cells are first exposed to mistranslation and then oxidative stress (induced in early or mid-exponential growth phase), the toxic effects of oxidative stress are less severe. Interestingly, pre-incubation with the lower Val concentration (0.75 mmol/L) showed even better, yet modest adaptive effect. However, more detailed research is needed to pinpoint conditions in which mistranslation of isoleucine codons confers beneficial effect under oxidative stress and to elucidate molecular mechanisms that enable this adaptation.