History-dependent physiological adaptation to lethal genetic modification under antibiotic exposure
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Evaluation Summary:
This paper presents results showing the temporal relationships between deletion of a resistance gene, introduction of antibiotic, and cell growth that are intriguing and novel. It will be of interest to researchers studying heterogeneity in antibiotic tolerance and the origins of drug resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Genetic modifications, such as gene deletion and mutations, could lead to significant changes in physiological states or even cell death. Bacterial cells can adapt to diverse external stresses, such as antibiotic exposure, but can they also adapt to detrimental genetic modification? To address this issue, we visualized the response of individual Escherichia coli cells to deletion of the antibiotic resistance gene under chloramphenicol (Cp) exposure, combining the light-inducible genetic recombination and microfluidic long-term single-cell tracking. We found that a significant fraction (∼40%) of resistance-gene-deleted cells demonstrated a gradual restoration of growth and stably proliferated under continuous Cp exposure without the resistance gene. Such physiological adaptation to genetic modification was not observed when the deletion was introduced in 10 hr or more advance before Cp exposure. Resistance gene deletion under Cp exposure disrupted the stoichiometric balance of ribosomal large and small subunit proteins (RplS and RpsB). However, the balance was gradually recovered in the cell lineages with restored growth. These results demonstrate that bacterial cells can adapt even to lethal genetic modifications by plastically gaining physiological resistance. However, the access to the resistance states is limited by the environmental histories and the timings of genetic modification.
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Author Response:
Reviewer #1 (Public Review):
[...]
Major points:
- I found the data on ribosomal protein stoichiometry to be somewhat unclear and had some questions about whether the results were statistically significant. Specific points: (a) In Fig. 5 it appears that growth-restored and growth-halted cells essentially have the same behavior, and pre-deleted cells are very similar. How are the growth-halted and pre-deleted cells growing in the presence of chloramphenicol? Also, why are the distributions essentially the same for all? (b) In Fig. 4E-F the behavior of both the growth-restored and growth-halted cells fluctuates a great deal. Are the differences between the two strains actually significant? It appears that the later timepoints (e.g. 65h) may start to diverge, but the experiments stop here so it is difficult conclude …
Author Response:
Reviewer #1 (Public Review):
[...]
Major points:
- I found the data on ribosomal protein stoichiometry to be somewhat unclear and had some questions about whether the results were statistically significant. Specific points: (a) In Fig. 5 it appears that growth-restored and growth-halted cells essentially have the same behavior, and pre-deleted cells are very similar. How are the growth-halted and pre-deleted cells growing in the presence of chloramphenicol? Also, why are the distributions essentially the same for all? (b) In Fig. 4E-F the behavior of both the growth-restored and growth-halted cells fluctuates a great deal. Are the differences between the two strains actually significant? It appears that the later timepoints (e.g. 65h) may start to diverge, but the experiments stop here so it is difficult conclude whether this is representative of the future or not.
Thank you for your remarks on the points where our data representation was confusing.
(a) In Figure 6A (previously, Fig. 5), we show the relations between the RplS-mCherry/RpsBmVenus fluorescence ratios and elongation rates for growth-restored cell lineages, growth-halted cell lineages, and pre-deleted cell lineages. The relations are indeed similar among the three types of cell lineages, but the distributions of the points are distinct. We now clarify this by showing the distributions of the points with density plots (new Figure 6B-D). As shown, the points for the growth-restored cell lineages are shifted toward the original ratio before deletion (=1) compared with those of growth-halted and pre-deleted cell lineages. This result is consistent with the restoration of both ribosomal proteins' stoichiometry and growth in the growth-restored cell lineages. We now explain this result in Results and show the density plots in Figure 6.
(b) As pointed out, the fluorescence ratios fluctuate significantly even for the same type of cell lineages. To examine the statistical difference of fluorescence ratio between growth-restored and growth-halted cell lineages, we calculated the p-value of the Mann Whitney U-test at each time point and plotted its transition (Figure 5-figure supplement 4). The result shows that the difference becomes evident and stable 37 hours after resistance gene deletion. We now refer to this in Results (and Figure 5-figure supplement 4).
- Growth is quantified in different ways in the manuscript, which make it difficult to compare different data and potentially masks information about cell division. In some cases, generation time is presented in minutes (e.g. Fig. 1E), in others generation time is presented in hours (e.g. Fig. 2I), and then the authors switch to elongation rates (e.g. Fig. 3B). The generation time vs. elongation rate could potentially mask behavior where cells are filamenting but not dividing. The differences in units makes it difficult to understand the growth impact on growth-restored cells. I gather from Fig. 4B that these growth-restored cells are barely growing?
- Do the growth-restored cells, which are very slow growing in chloramphenicol, return to normal growth after chloramphenicol has been removed?
- In the Discussion the authors describe this as a barely-tolerable state. This coupled with the use of a relatively modest antibiotic concentration (15 ug/ml) makes me wonder about how sensitive the findings are to antibiotic concentration. It would be interesting to see if the key effect observed in Fig. 2 is maintained at higher antibiotic concentrations.
- Single-cell resolution measurements are elegant and show the source of the survival, but cells growing in the mother machine do not compete with neighboring cells for resources. It could be interesting to repeat a key experiment in bulk cultures to show this or to speculate on how these results would look.
It is true that cells in the mother machine are unaffected by selection and can stay in the device even if they are slow-growing or non-growing. Since the growth of resistance-gene-deleted cells is significantly slower than that of non-deleted cells, it is conceivable that the fraction of resistance-gene-deleted cells decreases with time if they are competed with non-deleted cells. We indeed confirmed this by illuminating a population of YK0083 cells in a batch culture containing 15 µg/ml Cp by blue light for 30 min and by quantifying the fractions of cat-deleted and non-deleted cells (Figure 3-figure supplement 2B). The fraction of cat-deleted cells was 44.5% immediately after blue-light illumination but decreased to 13.4% in 6 hours. Therefore, the adaptation characterized in this study would be hardly recognized in batch culture experiments. We now mention this result in Results (and Figure 3-figure supplement 2B) and discuss the advantage of using the mother machine device to detect long-term adaptation phenomena that occur in slow-growing cell lineages.
Reviewer #2 (Public Review):
The authors addressed the question of whether bacteria can adapt physiologically to the deletion of an essential gene using an innovative combination of light inducible recombination, single-cell time-lapse microscopy, and bulk genetic analysis. The authors grew chloramphenicol (Cp) resistant E. coli cells in a mother machine microfluidic device. At a precisely controlled time recombination was triggered causing the loss of the resistance cassette, together with a linked fluorescent marker, in a fraction of the cells. As expected, cell division stopped after the loss of the resistance cassette, but remarkably, a sizable fraction of cells (~40%) could gradually resume growth, albeit at a reduced rate. The authors recovered offspring of these cells and used batch assays (MIC measurements and PCR) to confirm that they had lost their resistance cassette and where genetically susceptible to Cp; moreover, whole-genome sequencing confirmed that no other mutations had occurred, suggesting that the observed growth in Cp was due to physiological adaptation.
The authors subsequently showed that the timing of gene deletion was essential: if the deletion happens too long before Cp treatment cells cannot adapt anymore. They thus hypothesized that cells need at least a few copies of the Cat resistance protein to be able to physiologically adapt. Finally, the authors propose that the mechanism of adaptation could be related to the stoichiometric balance of ribosomal subunits. They used single cell reporters to show that cell growth correlates with the stoichiometric balance of RpsS and RpsB (part of 50S and 30S subunit respectively); cells that lose the resistance cassette become stoichiometric unbalanced; cells that can recover growth also recover their stoichiometric balance, suggesting that these two factors are at least correlated (though a causal relation was not shown).
Overall, the manuscript is clearly written, and most conclusions are well supported by the data (however, I have some concerns regarding the sample size of one of the essential control experiments, see below). I believe this paper makes an important conceptual and methodological contribution to the field: combining light inducible recombination with single cell microscopy opens promising avenues to explore the interplay of genetic and physiological adaptation in bacteria. This can give both insight in fundamental question regarding evolutionary dynamics, as well as more practical questions regarding e.g., antibiotic tolerance and resistance.
The authors finding that cells can keep growing in normally lethal concentration of Cp, despite being genetically susceptible to this antibiotic, is also very intriguing. However, it also raises many questions that are not addressed within the manuscript. Most importantly, the question remains open what the mechanism is behind the physiological adaptation (the link to stoichiometric balance of ribosomal subunits is purely correlation based and further experiments are needed to show a causative link). Moreover, many physiological questions remain unanswered: e.g., for how long can the adapted cells keep on growing in Cp? And how quickly is the physiological adaptation lost after Cp is removed? The manuscript thus raises many new questions that remain unanswered; however, I do not see this as a major limitation as the presented work is novel and interesting as it is, and it paves the way for follow-up work by the wider community.
We appreciate your positive evaluation. We agree that many important questions mentioned above still remain unanswered. We hope to address these issues in future studies.
In my opinion, the manuscript has only one main weakness: the authors conclusions critically depend on the analysis of cells recovered from the microfluidic devices: they use this data to conclude that all mCherry negative cells have lost the cat resistance cassette (Fig. S6), however I am a bit concerned with the small number (n=5) of cells on which this conclusion is based. The cells were recovered after growing them for 6h without Cp. As Cp is a bacteriostatic drug it is conceivable that during this period also some of the growth-halted cells resume growth. The recovered mCherry negative cells could thus come both from growth-halted and growth-recovered populations. In fact, if both groups recover at the same rate, there would be about a 10% chance (62.7%^5) that all 5 mCherry negative cells would be the offspring of growth-halted lineages. Potentially there could be a difference in genotype between the growth-halted and growth recovered populations (e.g. maybe the growth recovered only lost mCherry, not cat, while growth halted lost both). Without additional information there is thus not sufficient evidence to support the authors conclusion that all mCherry negative cells observed in the microfluidic device are also cat negative.
Thank you for pointing out an important issue. We addressed this through additional experiments and analyses. First, we confirmed the correspondence between the loss of mCherry fluorescence and the absence of cat resistance gene by additional colony PCR experiments (Figure 3-figure supplement 2A). We also quantified the fractions of regrowing cells for both growth-restored and growth-halted resistance-gene-deleted cells and found that the probability that all five mCherry negative cells were derived from growth-halted cells was 5.5%. We believe that these additional results support the conclusion more strongly.
Reviewer #3 (Public Review):
This study attempts to determine whether bacteria can "adapt to detrimental genetic modification." A E. coli strain with the CAT gene, which confers resistance to the bacteriostatic drug Chloramphenicol (Cp) was used. However, the gene was placed in such a way that the CAT gene can be removed from the genome on exposure to blue light. This is a creative way to alter the resistance levels. Although it appears that it does not work as well as chemical induction systems, there are many cases where chemical induction systems do not work. This optogenetic based method could be valuable to the field.
The conclusions reached in this paper, regarding the specific case of CAT gene loss shortly before Cp treatment, are well-supported by the data. But there are some issues to the relevance of these findings. It is already known that Cp is associated with adaptive resistance in wild-type bacteria - that is to say, the MIC is higher when the cells are exposed to a gradually-increasing concentration of the drug. This experimental control is a fancy way of probing adaptive resistance. From the perspective of the existing knowledge (i.e., Cp is associated with adaptive resistance), the findings are not particularly novel. As such, the statement "new insights into the emergence of drug-resistant bacterial and cancer cells" is not convincing. Some of these issues were mentioned (lines 222-227) but were not discussed in detail.
Thank you for calling our attention to the lack of discussion on potential relevance of the phenomenon characterized in this study to adaptive resistance. We now discuss this in detail, citing the references on adaptive resistance.
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Evaluation Summary:
This paper presents results showing the temporal relationships between deletion of a resistance gene, introduction of antibiotic, and cell growth that are intriguing and novel. It will be of interest to researchers studying heterogeneity in antibiotic tolerance and the origins of drug resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Studies of antibiotic tolerance at the single-cell level can tell us a great deal about bacterial physiology and hint at routes for the emergence of antibiotic resistance in clinical contexts. Here, Koganezawa et al. develop and use a light-activated recombinase system to excise the gene for chloramphenicol acetyltransferase at a precise time. They show that when E. coli cells experience this deletion after exposure to chloramphenicol, they are more likely to survive and resume growth under chloramphenicol treatment. First, the authors show that their system can cause the loss of the chloramphenicol gene in some cells, which they monitor via the loss of an attached fluorescent reporter. They then show that a fraction of cells that lose the chloramphenicol genes are still able to slowly divide while …
Reviewer #1 (Public Review):
Studies of antibiotic tolerance at the single-cell level can tell us a great deal about bacterial physiology and hint at routes for the emergence of antibiotic resistance in clinical contexts. Here, Koganezawa et al. develop and use a light-activated recombinase system to excise the gene for chloramphenicol acetyltransferase at a precise time. They show that when E. coli cells experience this deletion after exposure to chloramphenicol, they are more likely to survive and resume growth under chloramphenicol treatment. First, the authors show that their system can cause the loss of the chloramphenicol gene in some cells, which they monitor via the loss of an attached fluorescent reporter. They then show that a fraction of cells that lose the chloramphenicol genes are still able to slowly divide while experiencing continued chloramphenicol, and that this fraction decreases and disappears when the chloramphenicol gene is lost at increasing time intervals before chloramphenicol exposure. They also show that cells with a growth restoration phenotype have a partially-restored ratio of 50S/30S ribosomal subunit expression. Overall, the authors present an interesting system to explore the effects of history-dependence on bacterial antibiotic survival, and use a set of carefully-designed experiments and controls to show how gene loss can lead to diverse phenotypes within a bacterial population. I commend the authors on conducting a challenging study with many relevant controls. The ability to precisely delete genes and monitor cells in response is very interesting and the authors present interesting data about a window of time in which cells with the resistance genes deleted are able to survive. However, to fully support the author's conclusions there are several aspects of the manuscript that would benefit from additional data, alternative presentation formats, or further explanation, as detailed below.
Major points:
I found the data on ribosomal protein stoichiometry to be somewhat unclear and had some questions about whether the results were statistically significant. Specific points: (a) In Fig. 5 it appears that growth-restored and growth-halted cells essentially have the same behavior, and pre-deleted cells are very similar. How are the growth-halted and pre-deleted cells growing in the presence of chloramphenicol? Also, why are the distributions essentially the same for all? (b) In Fig. 4E-F the behavior of both the growth-restored and growth-halted cells fluctuates a great deal. Are the differences between the two strains actually significant? It appears that the later timepoints (e.g. 65h) may start to diverge, but the experiments stop here so it is difficult conclude whether this is representative of the future or not.
Growth is quantified in different ways in the manuscript, which make it difficult to compare different data and potentially masks information about cell division. In some cases, generation time is presented in minutes (e.g. Fig. 1E), in others generation time is presented in hours (e.g. Fig. 2I), and then the authors switch to elongation rates (e.g. Fig. 3B). The generation time vs. elongation rate could potentially mask behavior where cells are filamenting but not dividing. The differences in units makes it difficult to understand the growth impact on growth-restored cells. I gather from Fig. 4B that these growth-restored cells are barely growing?
Do the growth-restored cells, which are very slow growing in chloramphenicol, return to normal growth after chloramphenicol has been removed?
In the Discussion the authors describe this as a barely-tolerable state. This coupled with the use of a relatively modest antibiotic concentration (15 ug/ml) makes me wonder about how sensitive the findings are to antibiotic concentration. It would be interesting to see if the key effect observed in Fig. 2 is maintained at higher antibiotic concentrations.
Single-cell resolution measurements are elegant and show the source of the survival, but cells growing in the mother machine do not compete with neighboring cells for resources. It could be interesting to repeat a key experiment in bulk cultures to show this or to speculate on how these results would look.
-
Reviewer #2 (Public Review):
The authors addressed the question of whether bacteria can adapt physiologically to the deletion of an essential gene using an innovative combination of light inducible recombination, single-cell time-lapse microscopy, and bulk genetic analysis. The authors grew chloramphenicol (Cp) resistant E. coli cells in a mother machine microfluidic device. At a precisely controlled time recombination was triggered causing the loss of the resistance cassette, together with a linked fluorescent marker, in a fraction of the cells. As expected, cell division stopped after the loss of the resistance cassette, but remarkably, a sizable fraction of cells (~40%) could gradually resume growth, albeit at a reduced rate. The authors recovered offspring of these cells and used batch assays (MIC measurements and PCR) to confirm …
Reviewer #2 (Public Review):
The authors addressed the question of whether bacteria can adapt physiologically to the deletion of an essential gene using an innovative combination of light inducible recombination, single-cell time-lapse microscopy, and bulk genetic analysis. The authors grew chloramphenicol (Cp) resistant E. coli cells in a mother machine microfluidic device. At a precisely controlled time recombination was triggered causing the loss of the resistance cassette, together with a linked fluorescent marker, in a fraction of the cells. As expected, cell division stopped after the loss of the resistance cassette, but remarkably, a sizable fraction of cells (~40%) could gradually resume growth, albeit at a reduced rate. The authors recovered offspring of these cells and used batch assays (MIC measurements and PCR) to confirm that they had lost their resistance cassette and where genetically susceptible to Cp; moreover, whole-genome sequencing confirmed that no other mutations had occurred, suggesting that the observed growth in Cp was due to physiological adaptation.
The authors subsequently showed that the timing of gene deletion was essential: if the deletion happens too long before Cp treatment cells cannot adapt anymore. They thus hypothesized that cells need at least a few copies of the Cat resistance protein to be able to physiologically adapt. Finally, the authors propose that the mechanism of adaptation could be related to the stoichiometric balance of ribosomal subunits. They used single cell reporters to show that cell growth correlates with the stoichiometric balance of RpsS and RpsB (part of 50S and 30S subunit respectively); cells that lose the resistance cassette become stoichiometric unbalanced; cells that can recover growth also recover their stoichiometric balance, suggesting that these two factors are at least correlated (though a causal relation was not shown).
Overall, the manuscript is clearly written, and most conclusions are well supported by the data (however, I have some concerns regarding the sample size of one of the essential control experiments, see below). I believe this paper makes an important conceptual and methodological contribution to the field: combining light inducible recombination with single cell microscopy opens promising avenues to explore the interplay of genetic and physiological adaptation in bacteria. This can give both insight in fundamental question regarding evolutionary dynamics, as well as more practical questions regarding e.g., antibiotic tolerance and resistance.
The authors finding that cells can keep growing in normally lethal concentration of Cp, despite being genetically susceptible to this antibiotic, is also very intriguing. However, it also raises many questions that are not addressed within the manuscript. Most importantly, the question remains open what the mechanism is behind the physiological adaptation (the link to stoichiometric balance of ribosomal subunits is purely correlation based and further experiments are needed to show a causative link). Moreover, many physiological questions remain unanswered: e.g., for how long can the adapted cells keep on growing in Cp? And how quickly is the physiological adaptation lost after Cp is removed? The manuscript thus raises many new questions that remain unanswered; however, I do not see this as a major limitation as the presented work is novel and interesting as it is, and it paves the way for follow-up work by the wider community.
In my opinion, the manuscript has only one main weakness: the authors conclusions critically depend on the analysis of cells recovered from the microfluidic devices: they use this data to conclude that all mCherry negative cells have lost the cat resistance cassette (Fig. S6), however I am a bit concerned with the small number (n=5) of cells on which this conclusion is based. The cells were recovered after growing them for 6h without Cp. As Cp is a bacteriostatic drug it is conceivable that during this period also some of the growth-halted cells resume growth. The recovered mCherry negative cells could thus come both from growth-halted and growth-recovered populations. In fact, if both groups recover at the same rate, there would be about a 10% chance (62.7%^5) that all 5 mCherry negative cells would be the offspring of growth-halted lineages. Potentially there could be a difference in genotype between the growth-halted and growth recovered populations (e.g. maybe the growth recovered only lost mCherry, not cat, while growth halted lost both). Without additional information there is thus not sufficient evidence to support the authors conclusion that all mCherry negative cells observed in the microfluidic device are also cat negative.
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Reviewer #3 (Public Review):
This study attempts to determine whether bacteria can "adapt to detrimental genetic modification." A E. coli strain with the CAT gene, which confers resistance to the bacteriostatic drug Chloramphenicol (Cp) was used. However, the gene was placed in such a way that the CAT gene can be removed from the genome on exposure to blue light. This is a creative way to alter the resistance levels. Although it appears that it does not work as well as chemical induction systems, there are many cases where chemical induction systems do not work. This optogenetic based method could be valuable to the field.
The conclusions reached in this paper, regarding the specific case of CAT gene loss shortly before Cp treatment, are well-supported by the data. But there are some issues to the relevance of these findings. It is …
Reviewer #3 (Public Review):
This study attempts to determine whether bacteria can "adapt to detrimental genetic modification." A E. coli strain with the CAT gene, which confers resistance to the bacteriostatic drug Chloramphenicol (Cp) was used. However, the gene was placed in such a way that the CAT gene can be removed from the genome on exposure to blue light. This is a creative way to alter the resistance levels. Although it appears that it does not work as well as chemical induction systems, there are many cases where chemical induction systems do not work. This optogenetic based method could be valuable to the field.
The conclusions reached in this paper, regarding the specific case of CAT gene loss shortly before Cp treatment, are well-supported by the data. But there are some issues to the relevance of these findings. It is already known that Cp is associated with adaptive resistance in wild-type bacteria - that is to say, the MIC is higher when the cells are exposed to a gradually-increasing concentration of the drug. This experimental control is a fancy way of probing adaptive resistance. From the perspective of the existing knowledge (i.e., Cp is associated with adaptive resistance), the findings are not particularly novel. As such, the statement "new insights into the emergence of drug-resistant bacterial and cancer cells" is not convincing. Some of these issues were mentioned (lines 222-227) but were not discussed in detail.
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