NTH2 1271_1272delTA Gene Disruption Results in Salt Tolerance in Saccharomyces cerevisiae

Round 1

Reviewer 1 Report

English need to be improved. I recommend a native speaker or a specialized service to check the spelling.

Author Response

Reviewer 1.

 

English need to be improved. I recommend a native speaker or a specialized service to check the spelling.

Recommendation accepted. If accepted, we will have the document checked by a specialized service.

Reviewer 2 Report

The manuscript by Hernandez-Soto et al. reports the growth of wild-type and NTH1 or NTH2 mutants of S. cerevisiae cells in the presence of high salt concentrations, and the authors suggest that loss of NTH2 function may be a strategy for producing salt-resistant yeast for commercial uses. This manuscript is not suitable for publication in Fermentation because it does not represent enough of a scientific advance, has insufficient analysis of the mutant strains, and the rationale for some of the work reported is unclear. Specific reasons for my decision to reject the paper are given below.

 

It is not clear why the authors chose to use CRISPR-Cas9 to make mutations in the two genes when the goal appeared to knockout NTH1 or NTH2 function. The mutations made are referred to as knockout mutations, but they are really a very small deletion of 2 base pairs or an insertion of 1 base pair. It is a routine method to completely replace the coding sequence of a gene in yeast by homologous recombination-based transformation protocols, and it would have been easier to evaluate a complete gene deletion. While it is likely that these mutations fully disrupt enzyme activity of the proteins, there is no characterization of trehalose levels or trehalase activity in the yeast cells to determine how much activity is lost in each mutant. The description of generating the strains in the methods states that only one mutant strain for each strain was analyzed (section 2.4). It would be typical to look at 2 or 3 different transformants for each gene to be sure that effects observed are due to the mutation of interest and not due to off-target mutations produced through the transformation and CRISPR-Cas9 procedures. Considering that the authors mention that reference 13 did not find any effect of NTH2 on salt tolerance, it would have been very important to characterize trehalose levels and trehalase activity in control and stress conditions, or further evaluate the mutant in other ways to try to find a reason for the different result.

 

It is not clear why the yeast strains were analyzed by SEM and TEM. That is not a typical approach for comparing wild type and mutant yeast unless specific structural changes in the cell surface/shape or abundance/presence of organelles or other large intracellular structural features were expected in the mutants. The authors are extremely brief discussing these results and do not offer any specific explanation of why they might have expected to see differences between the cells using these methods.

 

Similarly, the modeling of the proteins is not necessary. Simply explaining what mutations were produced was sufficient, along with briefly discussing what parts of the proteins would be missing from the mutant versions. As noted above, characterizing the remaining enzyme activity in each mutant and trehalose levels would have been much more important than modeling the mutant proteins.

 

Also, the only result reported of the difference in growth in high salt in Figure 7 is difficult to evaluate. The spot dilutions plates do not include control plates to verify that similar amounts of cells were present in each spot. The data shows substantial growth of the nth2 mutant on 1.2 M NaCl, but throughout the paper the authors state the strain is resistant to 0.85 M NaCl. The graphs of the growth data are extremely small and very hard to read. The methods state that growth was measured over a range of 0 to 2.4 M NaCl, but the graphs only show 0 versus 0.85 M NaCl and do not show data for nth1 mutants (at least not in the upper panels for the current study).

 

The lower graph panels in Figure 7 are very confusing, and presented as if they show previously reported data from reference 28. The beginning paragraph of the Discussion also states that reference 28 previously showed that nth2 mutants were tolerant to salt. Based on that statement, the current study has simply confirmed a previous observation and not added anything new to our understanding of the topic. Reference 28 is a report about a publicly available database for examining and visualizing phenotypic data. From that paper, it is not clear what data the authors of the current paper are referring to. Data from another paper should not be included in a figure unless permission was granted by the previous authors or publisher. However, if the current authors used the database reported in reference 28 to find and visualize the data, that should be made clear in the manuscript. As is, the inclusion of those graphs is very confusing and seems to indicate that the salt tolerance of nth2 mutants was already observed and is just being confirmed in the current study.

Author Response

Reviewer 2

The manuscript by Hernandez-Soto et al. reports the growth of wild-type and NTH1 or NTH2 mutants of S. cerevisiae cells in the presence of high salt concentrations, and the authors suggest that loss of NTH2 function may be a strategy for producing salt-resistant yeast for commercial uses. This manuscript is not suitable for publication in Fermentation because it does not represent enough of a scientific advance, has insufficient analysis of the mutant strains, and the rationale for some of the work reported is unclear. Specific reasons for my decision to reject the paper are given below.

1. It is not clear why the authors chose to use CRISPR-Cas9 to make mutations in the two genes when the goal appeared to knockout NTH1 or NTH2 function. The mutations made are referred to as knockout mutations, but they are really a very small deletion of 2 base pairs or an insertion of 1 base pair. It is a routine method to completely replace the coding sequence of a gene in yeast by homologous recombination-based transformation protocols, and it would have been easier to evaluate a complete gene deletion.

Suggestion accepted.

-Knockout adjusted to loss-of-function/gene disruption.

- CRISPR use explained as follows.

Introduction.

Saccharomyces cerevisiae was used as our model organism for a proof-of-concept study to achieve salt-tolerant phenotypic traits using CRISPR/Cas9 editing and independent disruption of NTH2 compared to with wild type and NTH1 disruption. A CRISPR-derived mutant is considered conventional in many legal frameworks like Brazil, allowing further breeding commercial strains with no regulatory constraints [17]. We avoided other techniques such as homolog recombination because it could result in a genetically modified organism (GMO) limiting its industrial use.

 

2. While it is likely that these mutations fully disrupt enzyme activity of the proteins, there is no characterization of trehalose levels or trehalase activity in the yeast cells to determine how much activity is lost in each mutant.

Suggestion accepted and corrected as follows.

Discussion.

No important change of intracellular trehalose was previously reported when NTH2 is eliminated under osmotic NaCl stress [12,36]. However, in previous reports testing the relation of NTH1 and NTH2 with pressure tolerance, the trehalose content is slightly high but not statistically different in Δnth2 in stationary phase (Δnth2=316±66 μg/mg of protein, wt=257±47 μg mL^-1, Δnth1=519±80 μg mL^-1). Notably, Δnth2 acquired a barotolerance dismissed by the authors (Δnth2=5.0±2.0, wild type=3.4±1.0, Δnth1=0.3±0.09). Instead, the authors focus on Δnth1 sensitivity although having a higher concentration of trehalose [38]. High trehalose concentration can protect from pressure but requires hydrolysis mediated by NTH1 because it interferes with the reactivation of the cell [38,39]. A high trehalose concentration is insufficient for stress tolerance, but its correct use as an energy reservoir seems essential. Yeast cells subjected to 50 MPa of pressure results in immediately induction of TPS1 gene (at 0’,5’,10’,15’ was 2.41, 3.92, 4.15, 4.16) triggering trehalose synthesis, while NTH1 and NTH2 are induced primarily post-pressurisation (at 0’,5’,10’,15’ was NTH1=0.41, 2.07, 2.78, 3.14; NTH2=1.07, 2.23, 3.21, 3.73) [40].

 

3. The description of generating the strains in the methods states that only one mutant strain for each strain was analyzed (section 2.4). It would be typical to look at 2 or 3 different transformants for each gene to be sure that effects observed are due to the mutation of interest and not due to off-target mutations produced through the transformation and CRISPR-Cas9 procedures.

Suggestion accepted.

Selection and sequencing confirmation of mutants. Thirty (30) randomly selected strains were grown on YPD overnight media. Subsequently, the manufacturer's instructions obtained genomic DNA using the ReliaPrepTM gDNA Tissue Miniprep System (Promega, USA). Specific primers flanking the target sgRNA site of the NTH2 gene were designed and named nth2f: 5'-GCAAGAGGTATGGTGGAGCA-3' and nth2r: 5'-TTCAGCTAGCTCCTCCCAGT-3’ (Tm 55 °C; 539 bp); while NTH1 primers flanking the target sgRNA site were also designed and named nth1f: 5'-ACCCCCGGTTTACTAGCATTG-3' and nth1r: 5'-TAAGGTAACGCCGTGTTTCGA-3' (Tm 55 °C; 528 bp). Sanger sequencing of PCR products was performed in Macrogen at Maryland, USA, to confirm the mutation and absence of off-targets. We selected two isolates with the same mutation on the NTH2-disrupted gene and intact NTH1 gene, named Δnth2. Similarly, we selected two isolated with NTH1-disrupted gene and intact NTH2 gene, named Δnth1.

4. Considering that the authors mention that reference 13 did not find any effect of NTH2 on salt tolerance, it would have been very important to characterize trehalose levels and trehalase activity in control and stress conditions, or further evaluate the mutant in other ways to try to find a reason for the different result.

Suggestion accepted. We have limited access to our facilities to run accurate measures of trehalose content. However, we did not find differences in our preliminary data. We do not fully understand the behavior of the strain but already reported data in consistent with a slight increase of the sugar and provides clues on the tolerance as described next.

No important change of intracellular trehalose was previously reported when NTH2 is eliminated under osmotic NaCl stress [12,36]. However, in previous reports testing the relation of NTH1 and NTH2 with pressure tolerance, the trehalose content is slightly high but not statistically different in Δnth2 in stationary phase (Δnth2=316±66 μg/mg of protein, wt=257±47 μg mL^-1, Δnth1=519±80 μg mL^-1). Notably, Δnth2 acquired a barotolerance dismissed by the authors (Δnth2=5.0±2.0, wild type=3.4±1.0, Δnth1=0.3±0.09). Instead, the authors focus on Δnth1 sensitivity although having a higher concentration of trehalose [38]. High trehalose concentration can protect from pressure but requires hydrolysis mediated by NTH1 because it interferes with the reactivation of the cell [38,39]. A high trehalose concentration is insufficient for stress tolerance, but its correct use as an energy reservoir seems essential. Yeast cells subjected to 50 MPa of pressure results in immediately induction of TPS1 gene (at 0’,5’,10’,15’ was 2.41, 3.92, 4.15, 4.16) triggering trehalose synthesis, while NTH1 and NTH2 are induced primarily post-pressurisation (at 0’,5’,10’,15’ was NTH1=0.41, 2.07, 2.78, 3.14; NTH2=1.07, 2.23, 3.21, 3.73) [40].

 

 

5. It is not clear why the yeast strains were analyzed by SEM and TEM. That is not a typical approach for comparing wild type and mutant yeast unless specific structural changes in the cell surface/shape or abundance/presence of organelles or other large intracellular structural features were expected in the mutants. The authors are extremely brief discussing these results and do not offer any specific explanation of why they might have expected to see differences between the cells using these methods.

Accepted and corrected as follows.

Behavior of the Δnth2 strain under salinity stress: The Δnth2 strain has increased tolerance and can survive in high concentrations of NaCl (0.85M NaCl). We noted that Δnth2 strains were slightly smaller than the control under the light microscopy. Our first hypothesis was that salt-tolerant strains could have differences in their structures. However, the cells remained the same and had no statistical differences from the control under high osmolarity conditions. Yeast dimensions were determined from the scanning electron microscopy images (Figure 3A). The sizes of the cells remained statistically and phenotypically identical to the wild-type strain (Figure 3B).

The Δnth2 strain cells were also not different by transmission electron microscopy analysis. Organelles and structures such as vacuoles, nucleus, mitochondrion, cell membrane, and cell wall had no differences compared to the wild-type CEN.PK2-1C strain (Figure 4).

6. Similarly, the modeling of the proteins is not necessary. Simply explaining what mutations were produced was sufficient, along with briefly discussing what parts of the proteins would be missing from the mutant versions. As noted above, characterizing the remaining enzyme activity in each mutant and trehalose levels would have been much more important than modeling the mutant proteins.

Accepted and eliminated.

7. Also, the only result reported of the difference in growth in high salt in Figure 7 is difficult to evaluate. The spot dilutions plates do not include control plates to verify that similar amounts of cells were present in each spot. The data shows substantial growth of the nth2 mutant on 1.2 M NaCl, but throughout the paper the authors state the strain is resistant to 0.85 M NaCl. The graphs of the growth data are extremely small and very hard to read. The methods state that growth was measured over a range of 0 to 2.4 M NaCl, but the graphs only show 0 versus 0.85 M NaCl and do not show data for nth1 mutants (at least not in the upper panels for the current study). The lower graph panels in Figure 7 are very confusing, and presented as if they show previously reported data from reference 28. The beginning paragraph of the Discussion also states that reference 28 previously showed that nth2 mutants were tolerant to salt. Based on that statement, the current study has simply confirmed a previous observation and not added anything new to our understanding of the topic. Reference 28 is a report about a publicly available database for examining and visualizing phenotypic data. From that paper, it is not clear what data the authors of the current paper are referring to. Data from another paper should not be included in a figure unless permission was granted by the previous authors or publisher. However, if the current authors used the database reported in reference 28 to find and visualize the data, that should be made clear in the manuscript. As is, the inclusion of those graphs is very confusing and seems to indicate that the salt tolerance of nth2 mutants was already observed and is just being confirmed in the current study.

 

Accepted and corrected. We focus on NTH2 behavior.

The Δnth2 strain was viable in a maximum concentration of 0.85 M NaCl and gave a standard growth curve and an average growth rate of 0.2327 ± 0.0057 h^-1 (Figure 5, Figure 6). When statistically analyzing the specific growth rates (p<0.05), the Δnth2 strain under stress conditions had a growth rate of 0.2179 ± 0.0061 h^-1 and behaved the same as the wild-type strain under normal conditions 0.2255 ± 0.0037 h^-1. The behavior of the wild-type strain in NaCl solution presented a significant decrease in the activity of 0.1580 ± 0.0009 h^-1.

We expected a slight salt tolerance because of the neglected reported activity of NTH2. The latter was reasonable because the strain still had functional NTH1 enzymes that metabolize trehalose. However, the data obtained showed that the Δnth2 strains were superiorly tolerant. The Δnth2 strains had an average growth curve in a 0.85M NaCl liquid medium, with three different strains having the same mutation and behavior. Instead, the wild type had a slower growth curve (Figure 5).

Figure 5. Growth curves of S. cerevisiae Δnth2 strains in the presence and absence of osmotic stress (0.85 M NaCl) by indirect measurements of optical density at 600 nm after 20 h of growth under stress. The curve was built with the mean of 4 independent samples per hour for each condition.

We mutated the homolog gen NTH1 resulting in Δnth1 strains to compare it with Δnth2. The Δnth1 strains were viable on 0.85 M NaCl and showed a slight tolerance to 1.2M NaCl when growing on agar plate-based comparison after 2weeks (Figure 6). However, we could not detect such Δnth1 tolerance when growing the mutant in 0.85 M NaCl liquid media (data not presented).

Figure 6. Agar plate comparison of yeast strains: Δnth1, Δnth2, and wildtype after 2weeks of growth under 0, 0.85 M and 1.2M NaCl stress. Note serial dilutions of yeast starting in OD₆₀₀= 1

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

I enjoyed reading this paper, interesting use of CRISPR-Cas9 technology. My question for the authors is, what is the end application of such a mutated microorganism? COuld they cause problems for the wild-type strain? I suggest authors address this in a paragraph if possible.

Author Response

Reviewer 3

 

I enjoyed reading this paper, interesting use of CRISPR-Cas9 technology. My question for the authors is, what is the end application of such a mutated microorganism? COuld they cause problems for the wild-type strain? I suggest authors address this in a paragraph if possible

 

Recommendation accepted.

 

NTH2 disrupted mutants, on the other hand, can mobilize and use trehalose. Its mutation results in an increased acid trehalase activity [19], meaning that the metabolic stability of the strain is not compromised. In addition, no significant change of intracellular trehalose occurs when NTH2 is eliminated under osmotic NaCl stress [12,37]. The latter also means trehalose negatively affects growth, for overaccumulation is unfeasible [16]. Interestingly, in Cryptococcus neoformans, the disruption of NTH2 alone increased the survival ability of the yeast, but the deletion of NTH1-NTH2 was negative for the microorganism [41].

Breeding industrial yeast can result in cost-effective or reductions in fermentation. In the case of stress tolerance traits, yeast is constantly exposed to ethanol toxicity, oxidative stress, temperature stress, and osmotic stress, diminishing its capacity to produce ethanol [42,43]. Our data shows that the osmotic tolerance of an NTH2 disruption strain mediated by CRISPR is superior and could represent a solution for the fermentation industry without compromising its metabolism, phenotype, or behavior [44,45].

Author Response File: Author Response.pdf

Reviewer 4 Report

According to previous studies especially references 12 and 18 which have done more extensive work and more detailed study on the two genes NTH2 or NTH1, there is no novelty in the present work. CRISPR-Cas9 as a tool has been only used to knock down the genes in this study. It would be noted that the use of new methods should lead to novelties in results otherwise it has no value.

Author Response

Reviewer

According to previous studies especially references 12 and 18 which have done more extensive work and more detailed study on the two genes NTH2 or NTH1, there is no novelty in the present work. CRISPR-Cas9 as a tool has been only used to knock down the genes in this study. It would be noted that the use of new methods should lead to novelties in results otherwise it has no value.

 

Suggestion Accepted. We adjusted the paper accordingly. For example,

 

Introduction

Saccharomyces cerevisiae was used as our model organism for a proof-of-concept study to achieve salt-tolerant phenotypic traits using CRISPR/Cas9 editing and independent disruption of NTH2 compared to with wild type and NTH1 disruption. A CRISPR-derived mutant is considered conventional in many legal frameworks like Brazil, allowing further breeding commercial strains with no regulatory constraints [17]. We avoided other techniques such as homolog recombination because it could result in a genetically modified organism (GMO) limiting its industrial use.

 

In this article, we report a CRISPR/Cas9-specific NTH2 disruption in S. cerevisiae that results in the capacity of the yeast to grow in 0.85 M NaCl and tolerate 1.2M NaCl in comparison with non-mutant and Δnth1 strains. We propose that disrupting NTH2 alone in yeast can trigger stress tolerance. To our knowledge, there is no formal report linking the disruption of NTH2 alone to stress tolerance in S. cerevisiae. The knowledge generated herein is potentially valuable to improve industrial yeast or serve as a model to develop osmotic stress tolerance in other organisms using a similar rationale.

 

Discussion

We foresee the disruption of NTH2 to provide stress tolerance in an industrial strain because NTH1 can provide the metabolic equilibrium as described next. NTH1 and NTH2 are required and regulated for fueling growth. NTH1 is phosphorylated by Cdk1(S66) and PKA1 (S20, S21, S60, S83) to be activated, and is required for fueling biosynthesis during S, G2, M [34]. NTH2 contains an N terminal phosphorylation region (R49, S52, R109, S112) and is expressed at a high level in stationary after glucose exhaustion [15]. NTH2 and NTH1 are downregulated at the exponential phase and have a higher expression at the stationary phase [19,35]. The presence of salinity stress causes trehalose accumulation in S. cerevisiae and higher ethanol osmotolerance [13,25]. Heat stress (40C), CuSO₄, NaAsO₂, H₂O₂, cycloheximide (CHX) but not NaCl (1.5M) trigger the expression of NTH1, and in practice, its disruption is unrequired for salt tolerance [36]. It is also known that strains with NTH2 disruption, previously named YBR0106, grow normal in YEP glycerol and were associated with increased sensitivity against heat shock at 50°C [14], while Δnth1 grows poorly in YEP glycerol and cannot mobilize endogenous trehalose [24]. NTH1 disruption improves stress tolerance, as previously reported [22,37]. However, Δnth1 may not be useful for industry because these mutants cannot hydrolyze trehalose after returning from heat stress temperature of 40°C to an average growth temperature of 30°C [15].

No important change of intracellular trehalose was previously reported when NTH2 is eliminated under osmotic NaCl stress [12,36]. However, in previous reports testing the relation of NTH1 and NTH2 with pressure tolerance, the trehalose content is slightly high but not statistically different in Δnth2 in stationary phase (Δnth2=316±66 μg/mg of protein, wt=257±47 μg mL^-1, Δnth1=519±80 μg mL^-1). Notably, Δnth2 acquired a barotolerance dismissed by the authors (Δnth2=5.0±2.0, wild type=3.4±1.0, Δnth1=0.3±0.09). Instead, the authors focus on Δnth1 sensitivity although having a higher concentration of trehalose [38]. High trehalose concentration can protect from pressure but requires hydrolysis mediated by NTH1 because it interferes with the reactivation of the cell [38,39]. A high trehalose concentration is insufficient for stress tolerance, but its correct use as an energy reservoir seems essential. Yeast cells subjected to 50 MPa of pressure results in immediately induction of TPS1 gene (at 0’,5’,10’,15’ was 2.41, 3.92, 4.15, 4.16) triggering trehalose synthesis, while NTH1 and NTH2 are induced primarily post-pressurisation (at 0’,5’,10’,15’ was NTH1=0.41, 2.07, 2.78, 3.14; NTH2=1.07, 2.23, 3.21, 3.73) [40].

NTH2 disrupted mutants, on the other hand, can mobilize and use trehalose. Its mutation results in an increased acid trehalase activity [19], meaning that the metabolic stability of the strain is not compromised. In addition, no significant change of intracellular trehalose occurs when NTH2 is eliminated under osmotic NaCl stress [12,37]. The latter also means trehalose negatively affects growth, for overaccumulation is unfeasible [16]. Interestingly, in Cryptococcus neoformans, the disruption of NTH2 alone increased the survival ability of the yeast, but the deletion of NTH1-NTH2 was negative for the microorganism [41].

Breeding industrial yeast can result in cost-effective or reductions in fermentation. In the case of stress tolerance traits, yeast is constantly exposed to ethanol toxicity, oxidative stress, temperature stress, and osmotic stress, diminishing its capacity to produce ethanol [42,43]. Our data also shows that the osmotic tolerance of an NTH2 disruption strain mediated by CRISPR is superior and could represent a solution for the fermentation industry without compromising its metabolism, phenotype, or behavior [44,45].

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The revised manuscript by Hernandez et al. on the salt tolerance of S. cerevisiae nth2 mutants has been somewhat improved from the original, but would need further substantial revisions to be considered acceptable for publication in Fermentation. Improvements include providing the reasoning for using CRISPR-Cas9 to generate the mutants, removing the protein modeling, better explaining the reasoning for using SEM, better graphs showing the salt tolerance data, and including the control serial dilutions for the spot-dilution test of salt tolerance. However, the manuscript is still only a limited advance in our knowledge due to limited characterization of the mutants and the absence of data providing possible reasons for the salt tolerance or why the results differ from prior reports that suggested that nth2 mutants did not have improved salt resistance. The authors would still need to address some major concerns for the manuscript to be publishable:

 

1. There needs to be some work to further investigate the salt stress of the nth2 disruption mutants. The authors state in the Discussion that their results may differ from a prior report (ref 13) due to differences in methodology. This is not sufficient to address the difference. The authors note that the prior work only examined the strains after 6 hours of growth, but the authors show that nth2 mutants and wild type are growing at very different rates after 6 h in Figure 5. One way to better address this is determining trehalose levels or trehalase activity in the wild type and mutant cells to see if the salt tolerance can be explained by changes trehalose or trehalase. The authors responded that they are unlikely to be able to quantify trehalose in cells. A second way to better address the difference is to make a standard deletion mutant of NTH2 using homologous recombination to determine if a knockout has a different phenotype than the disruption mutants. The authors explain that they used CRISPR-Cas9 so that they would not generate GMO yeast, but my suggestion is just to make a full deletion of NTH2 by homologous recombination for comparison purposes. While the disruption is likely resulting in nonfunctional Nth2 protein in yeast, there is always a possibility that a partial protein is being produced to some extent and producing a phenotype different than a full knockout mutant. Including mutants with a full deletion of the gene in the study would show if the phenotype is particular to the disruption mutation that the authors analyzed. If the deletion mutant does not show salt tolerance, that could explain the different results from the prior publication. If the deletion mutant does show salt tolerance, that would strengthen the authors’ conclusion that loss of NTH2 results in salt stress by having tested a different set of mutant strains.

 

2. There is limited analysis of the salt tolerance. The original manuscript had mentioned testing growth over a wide range of salt concentrations, but Figure 5 shows growth rates only for one concentration of salt, and no data are shown for nth1 mutants. The spot dilutions are done with two concentrations of salt, but those data raise the question of why nth2 mutants would not grow in 1.2 M NaCl in the automated growth assay if they grow so well on the petri plate. It would be better to show growth over a range of salt concentrations for wild type and nth2 mutants to show at what point wild type growth becomes slowed by the salt concentration and at what point the growth of nth2 mutants is slowed by the salt concentration. The authors point out in the text that nth2 mutants are growing at a wild type rate in 0.85 M NaCl. At what concentration would their growth slow down? Graphs showing growth at several different informative concentrations of NaCl (such as points at which wild type growth slows and at which nth2 mutant growth slows) would better characterize the salt tolerance. These assays should be straightforward for the authors using the automated procedure described in the manuscript. There should also be at least some data shown for nth1 mutants to provide support for the interpretation that they are not different from wild type.

 

3. If the authors cannot characterize trehalose and trehalase, as suggested in point 1 above, then they should do something more to add to our understanding of the nth2 mutants. There needs to be more characterization of the mutants for this work to provide more of an advance in the field. One possibility would be to determine whether the nth2 mutants are also resistant to a couple other common stresses (such as ethanol, or some other similar stress). That would show whether the mutants have a general increase in stress resistance, or whether there is only a very specific change in stress tolerance. Showing resistance to a variety of stresses would make nth2 mutants more relevant to a variety of industrial applications, while showing specificity for resistance to salt but not for other stresses would provide more information for understanding what might be happening in the mutants. Overall, there needs to be some more work with the mutants to better understand what is going on in the cells.

 

4. It is better explained how the SEM is being used to examine cell size. However, the TEM work still does not seem that worthwhile to show. The authors state that there are no visible differences in the mutant and wild type cells, but is that based on quantifying and measuring the average sizes of multiple organelles or just an overall visual inspection? For the SEM, there is a clear prediction that is being tested about cell size, but for TEM, it is not clear what the predicted changes would be. It seems better to not show the data, rather than to present a careful analysis of how organelles are the same in the wild type and mutant cells, unless there was a specific prediction of what organelle or cellular structure would be likely to change in the cells. The authors could potentially refer to this analysis having been done in the Results as data not shown.

 

 

Additional minor points

 

The title emphasizes CRISPR-Cas9, but it seems that the main message of the paper is that NTH2 disruption results in salt tolerance. CRISPR-Cas9 just happens to be the way the authors chose to make a disruption but is not critical to the salt tolerance.

 

Use of the ∆ symbol for naming the mutants is still confusing, as that is usually the symbol used for a deletion/knockout. Considering that these are disruptions due to small insertions, it would be better to not use the symbol that is used for a complete deletion.

 

Line 144 refers to growing on PDA for 48 h, but I did not see any explanation of what PDA stands for in the methods.

 

In the methods, the authors discuss selecting two isolates for ∆nth1 and ∆nth2. In the Results, though, they refer to selecting three mutant strains for each gene. That should be clarified.

 

In Figure 3A, many wild type cells in 0.85 M NaCl look larger than the cells shown for the 0 M NaCl image. Are the images reversed or are those cells not representative of the overall sizes?

 

The numbers on the axes in Figure 5 are much too small.

 

The authors could elaborate a little on the predictive model that suggested salt tolerance for NTH2 deletion in the Discussion (ref 30), which would be more relevant for considering the main message of their work, as opposed to spending a whole paragraph further discussing that they obtained the expected types of mutants from using CRISPR/Cas9.

Author Response

The revised manuscript by Hernandez et al. on the salt tolerance of S. cerevisiae nth2 mutants has been somewhat improved from the original, but would need further substantial revisions to be considered acceptable for publication in Fermentation. Improvements include providing the reasoning for using CRISPR-Cas9 to generate the mutants, removing the protein modeling, better explaining the reasoning for using SEM, better graphs showing the salt tolerance data, and including the control serial dilutions for the spot-dilution test of salt tolerance. However, the manuscript is still only a limited advance in our knowledge due to limited characterization of the mutants and the absence of data providing possible reasons for the salt tolerance or why the results differ from prior reports that suggested that nth2 mutants did not have improved salt resistance. The authors would still need to address some major concerns for the manuscript to be publishable:

We run a test with salt and no salt stress to measure trehalose content. The content of trehalose was not different from the control in NTH2 1271_1272delTA. We adjusted the article in content and proposals.

1. There needs to be some work to further investigate the salt stress of the nth2 disruption mutants. The authors state in the Discussion that their results may differ from a prior report (ref 13) due to differences in methodology. This is not sufficient to address the difference. The authors note that the prior work only examined the strains after 6 hours of growth, but the authors show that nth2 mutants and wild type are growing at very different rates after 6 h in Figure 5. One way to better address this is determining trehalose levels or trehalase activity in the wild type and mutant cells to see if the salt tolerance can be explained by changes trehalose or trehalase. The authors responded that they are unlikely to be able to quantify trehalose in cells. A second way to better address the difference is to make a standard deletion mutant of NTH2 using homologous recombination to determine if a knockout has a different phenotype than the disruption mutants. The authors explain that they used CRISPR-Cas9 so that they would not generate GMO yeast, but my suggestion is just to make a full deletion of NTH2 by homologous recombination for comparison purposes. While the disruption is likely resulting in nonfunctional Nth2 protein in yeast, there is always a possibility that a partial protein is being produced to some extent and producing a phenotype different than a full knockout mutant. Including mutants with a full deletion of the gene in the study would show if the phenotype is particular to the disruption mutation that the authors analyzed. If the deletion mutant does not show salt tolerance, that could explain the different results from the prior publication. If the deletion mutant does show salt tolerance, that would strengthen the authors’ conclusion that loss of NTH2 results in salt stress by having tested a different set of mutant strains.

 

We are trying to access Euroscarf strains http://www.euroscarf.de/search.php?search=nth2  kanMX4 NTH2 disruption strains. However, it seems to be clueless since the website's growth models of such Euroscarf yeast strains exist at http://prophecy.lundberg.gu.se/Qsearch.aspx?ORF=nth2  although never reported in the scientific literature.

We discover the database and behavior by serendipity when exploring math models to create our growth curves but found no article, review, literature mentioning such behavior.

 

1. There is limited analysis of the salt tolerance. The original manuscript had mentioned testing growth over a wide range of salt concentrations, but Figure 5 shows growth rates only for one concentration of salt, and no data are shown for nth1 mutants. The spot dilutions are done with two concentrations of salt, but those data raise the question of why nth2 mutants would not grow in 1.2 M NaCl in the automated growth assay if they grow so well on the petri plate. It would be better to show growth over a range of salt concentrations for wild type and nth2 mutants to show at what point wild type growth becomes slowed by the salt concentration and at what point the growth of nth2 mutants is slowed by the salt concentration. The authors point out in the text that nth2 mutants are growing at a wild type rate in 0.85 M NaCl. At what concentration would their growth slow down? Graphs showing growth at several different informative concentrations of NaCl (such as points at which wild type growth slows and at which nth2 mutant growth slows) would better characterize the salt tolerance. These assays should be straightforward for the authors using the automated procedure described in the manuscript. There should also be at least some data shown for nth1 mutants to provide support for the interpretation that they are not different from wild type.

 

Data included. It grows well on 1.2M but takes three days (60h) on solid media for the NTH2 mutant, and a week (7 days, 168h) for the control and NTH1 mutants to grow.  NTH1 trehalose content was analyzed. We can eliminate NTH1 data in case of necessary, since we couldn’t find any tolerance in the nth1 mutant.

 

 

1. If the authors cannot characterize trehalose and trehalase, as suggested in point 1 above, then they should do something more to add to our understanding of the nth2 mutants. There needs to be more characterization of the mutants for this work to provide more of an advance in the field. One possibility would be to determine whether the nth2 mutants are also resistant to a couple other common stresses (such as ethanol, or some other similar stress). That would show whether the mutants have a general increase in stress resistance, or whether there is only a very specific change in stress tolerance. Showing resistance to a variety of stresses would make nth2 mutants more relevant to a variety of industrial applications, while showing specificity for resistance to salt but not for other stresses would provide more information for understanding what might be happening in the mutants. Overall, there needs to be some more work with the mutants to better understand what is going on in the cells.

 Done. Data analyzed. We are validating how to perform other stress test (ethanol and saccharose osmotic pressure) in case of acceptance.

1. It is better explained how the SEM is being used to examine cell size. However, the TEM work still does not seem that worthwhile to show. The authors state that there are no visible differences in the mutant and wild type cells, but is that based on quantifying and measuring the average sizes of multiple organelles or just an overall visual inspection? For the SEM, there is a clear prediction that is being tested about cell size, but for TEM, it is not clear what the predicted changes would be. It seems better to not show the data, rather than to present a careful analysis of how organelles are the same in the wild type and mutant cells, unless there was a specific prediction of what organelle or cellular structure would be likely to change in the cells. The authors could potentially refer to this analysis having been done in the Results as data not shown.

Done. We performed TEM to validate no organelle fragmentation that is linked to salt susceptibility. The picture will be added to supplementary materials.

 

Additional minor points

 

The title emphasizes CRISPR-Cas9, but it seems that the main message of the paper is that NTH2 disruption results in salt tolerance. CRISPR-Cas9 just happens to be the way the authors chose to make a disruption but is not critical to the salt tolerance.

Done. NTH2 1271_1272delTA gene disruption results in salt tolerance in Saccharomyces cerevisiae

Use of the ∆ symbol for naming the mutants is still confusing, as that is usually the symbol used for a deletion/knockout. Considering that these are disruptions due to small insertions, it would be better to not use the symbol that is used for a complete deletion.

Adjusted to show the specific mutation instead.

Line 144 refers to growing on PDA for 48 h, but I did not see any explanation of what PDA stands for in the methods.

Done. Potato Dextrose.

In the methods, the authors discuss selecting two isolates for ∆nth1 and ∆nth2. In the Results, though, they refer to selecting three mutant strains for each gene. That should be clarified.

Done. It is two isolates.

In Figure 3A, many wild type cells in 0.85 M NaCl look larger than the cells shown for the 0 M NaCl image. Are the images reversed or are those cells not representative of the overall sizes?

The picture does not represent the overall sizes. We did the statistical analysis with multiple photos but there is no difference. It is just the best light – contrast picture selected by the technical person.

The numbers on the axes in Figure 5 are much too small.

Adjusted.

The authors could elaborate a little on the predictive model that suggested salt tolerance for NTH2 deletion in the Discussion (ref 30), which would be more relevant for considering the main message of their work, as opposed to spending a whole paragraph further discussing that they obtained the expected types of mutants from using CRISPR/Cas9.

Done as follows. Interestingly, in Cryptococcus neoformans, the disruption of NTH2 alone increased the survival ability of the yeast, but the deletion of NTH1-NTH2 was negative for the microorganism [41]. Similarly, a database of yeast mutants growth modeling done with kanMX4 interrupting NTH2 in haploid BY4741 background predicts tolerance to salt stress, such as our results [30].

 

Reviewer 4 Report

The authors did not answer my questions exactly!

Author Response

Trehalose itself is not enough to provide osmotolerance. For instance, deletion of NTH1 (Δnth1) does have a higher trehalose concentration but is not osmotolerant tolerant [Ref. 38].

In this case, our mutation of NTH2 provides tolerance to 0.85m of NaCl.

The novelty is that deletion NTH2 was reported to have a neglected influence on salt tolerance, contrary to the mutant disruption we obtained.

It is also novel that NTH2 1271_1272delTA mutation could be induced on commercial strains resulting in Non-GMO strains.