Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria
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Evaluation Summary:
The work supports the hypothesis that novel specific enymes evolved to modify metabolic pathways, allowing phototrophs to shift from growth under photoautotrophic and photomixotrophic growth conditions.
(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. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD + . Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.
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Author Response
Reviewer #1 (Public Review):
In Wang et al., the authors investigate issues related to the relative proportion of flux for the enzymatic decarboxylation of pyruvate between PDH (pyruvate dehydrogenase) and PFOR (pyruvate-ferredoxin oxoreductase) in the model organism Synechococystis. The manuscript provides evidence that PDH becomes increasingly inactivated by a high ratio of NADH:NAD+ as well as evidence to suggest that PFOR is transcribed and remains intact under aerobic conditions. The authors put forward the theory that both PDH and PFOR are functionally active routes for pyruvate decarboxylation under aerobic conditions, whereas PFOR has previously been assumed to be inactive under growth conditions containing oxygen. This distinction is particularly highlighted by conditions where Synechocystis is grown …
Author Response
Reviewer #1 (Public Review):
In Wang et al., the authors investigate issues related to the relative proportion of flux for the enzymatic decarboxylation of pyruvate between PDH (pyruvate dehydrogenase) and PFOR (pyruvate-ferredoxin oxoreductase) in the model organism Synechococystis. The manuscript provides evidence that PDH becomes increasingly inactivated by a high ratio of NADH:NAD+ as well as evidence to suggest that PFOR is transcribed and remains intact under aerobic conditions. The authors put forward the theory that both PDH and PFOR are functionally active routes for pyruvate decarboxylation under aerobic conditions, whereas PFOR has previously been assumed to be inactive under growth conditions containing oxygen. This distinction is particularly highlighted by conditions where Synechocystis is grown photomixotrophically - and where the NADH:NAD+ pool may be relatively over-reduced because of two parallel inputs of reductant (water-splitting at PSII and catabolism of glucose). The authors examine growth under photoautotrophic and photomixotrophic conditions for a number of relevant mutants including members of the ferredoxin/flavodoxin family, PFOR, and NDH-1 complex subunits.
The theory put forward in this manuscript is of general interest regarding electron flux through the combined electron transport chain (photosynthetic + respiratory) of cyanobacteria. The authors further broaden the potential audience for the manuscript by elaborating on the potential significance of these results in the context of a switch from PFOR (ancestral) to PDH (oxygenic/modern).
Comments:
Generally, theories put forward in this manuscript are intriguing and have a number of potential implications for understanding electron flux and regulation of central metabolic processes in photosynthetic microorganisms. If these theories are supported and become more generally adopted, they would have significant impact on the understanding of the regulation of central carbon metabolism in cyanobacteria. That said (due in no small part to the complexity of some of these pathways), the evidence provided to support the hypotheses is indirect in many instances. In some cases, there is a pairing of indirect data with broad statements that can come across as over-reach. These problems can be somewhat exacerbated by an unclear organization at parts of the Discussion, a lack of succinctly defined claims, and numerous typographical considerations.
Thank you very much for this point. We now reorganized the discussion and overhauled it completely. It starts with aspects that are best supported by our data. We then added two sentences to stress that the following lines include hypothetical considerations that are meant as thought-provoking impulses. We hope that thereby over-reach is prevented.
Major considerations:
A major component of the proposed theories in this manuscript rest upon the assumption that PFOR is an active enzyme under highly aerobic conditions: this claim is never directly demonstrated.
This is true. We could show though that PFOR of Synechocystis is in constrast to most bacterial PFORs stable in the presence of oxygen. However, as stated likewise for the oxygen stable PFOR of the obligate aerobe Sulfolobus acidocaldarius (3), and PFOR from E. coli, which was recently shown to contribute to metabolism in the presence of oxygen in vivo (1) we as well had to remove oxygen for enzyme acitivty in vitro. This point is discussed frankly.
Indirect evidence of altered growth of pfor mutants, increased repression of PDH, and the higher NADH:NAD+ ratio under photomixotrophic conditions is in general alignment with this theory. However, while deletion of pfor does indeed result in altered growth dynamics in Synechocystis under periods of photomixotrophy, the alterations do not entirely align with the idea that this pathway is critical for rapid growth under aerobic conditions. For instance, pfor and most of the highlighted mutants (fdx 3, fdx 9, isiB) presented in Figure 3 show the greatest defects in their OD after reaching stationary phase (more rapid decline in OD on/after Day 6) relative to WT. This doesn't align as nicely with the highest NADH:NAD+ seen in Days 3-5 (which is also specifically called out: e.g., Line 146, Supplemental Figure S8).
We are very cautious to compare growth experiments day by day. This is due to the fact that the growth behaviour of WT and mutants differ between experiments. We therefore repeat these experiments in several independent experiments including at least three replicates and show the data of typical growth experiments. In the case of the shown growth behaviour of WT and pfor and the NADH/NAD+ ratios under photoautotrophic and photomixotrophic conditions shown in figure 1, NADH/NAD+ ratios were determined in exactly those cultures for which growth data are shown. It is therefore legitimate to directly compare these results day by day. However, we did not determine the NADH/NAD+ ratios of the cultures shown in Fig. 3. The rise in NADH might have started with a delay here.
In this context, the deletion of F-GOGAT is much more convincing in it's severity and timing, yet for this mutation to have a more severe phenotype is unexpected if PFOR is one of the primary/sole electron donors to the ferredoxin pool from glucose utilization as proposed (i.e., stated differently, F-GOGAT is only one of the enzymes downstream of ferrodoxin and might be expected to have a more subtle phenotype in comparison to the KO of PFOR if that is a primary source for electrons to ferredoxin under photoheterotrophic conditions).
F-GOGAT requires reduced ferredoxin which can be provided by PFOR and in addition also by PSI. As electrons from glucose oxidation can be fed via photosynthetic complex I into the PQ-pool they will eventually arrive at PSI (Fig. 3C) where ferredoxin can be reduced and transfer electrons to F-GOGAT. However, to get a truly complete picture of the situation several issues will have to be addressed in the future: we do not know which of the low abundant ferredoxins as well as high abundant ferredoxin 1 interact with PSI, F-GOGAT, PFOR and photosynthetic complex I. It would be furthermore helpful to know all midpoint potentials of the different ferredoxins. Without this information it might be too much to ask for a simple interpretation.
A central tenant of the argument put forward on the evolutionary importance of using either PFOR vs. PDH is the conservation of extra free energy by the former reaction. However, additional information on the ferredoxin paralog(s) that accept electrons from PFOR is necessary to evaluate these claims. Based on the data within these manuscripts, Fdx3, Fdx9, and IsiB have the strongest links to PFOR: though the authors do take care to never state directly that they have evidence that these are the acceptors in vivo. Given the variability in the midpoint potentials of different ferredoxins, some ferredoxin acceptors may better conserve the free energy in pyruvate, while others may actually be more 'wasteful' than NAD+ as the acceptor through PDH. Unfortunately, the midpoint potentials for Fdx3, Fdx9, and IsiB are unknown or not stated in this manuscript. It is therefore unclear what ferredoxin is being used as the reference point for conservation of Gibbs free energy in Figure 4C and referenced multiple times in the text.
We agree that it would be great if we already knew the redox potentials of all the ferredoxins involved. We are currently working on this issue. All that we know for now is that the redox potentials of ferredoxins lay between -240 mV to -680 mV whereas the redox potential is around -320 mV for NAD(P)H/NAD(P)+. Unpublished data that require further validation reveal that the redox potential of Fdx9 is definitely more negative than the redox potential of Fdx1 (-412 mV) in Synechocystis and is thereby clearly more negative that -320 mV. However, as these data require further validation, we did not name numbers. In addition, interaction studies on PFOR and low abundant ferredoxins are planed and preparations are in progress.
Finally, the measurements of NADH:NAD+ (most prominently used for measurements in Fig 1B) utilized kits that require multiple, long centrifugation steps in the dark prior to assaying this rapidly exchanging pool. While it appears that the authors were able to get reproducible results with these kits, it is difficult to interpret what the increase in relative NADH levels in glucose-fed cells means given that 10+ minutes of incubation in the dark and/or changing temperatures elapsed after the cyanobacteria were removed from the incubator before the NADH:NAD ratio was assessed. While it superficially makes logical sense that the cytosol would be over-reduced when illuminated and under glucose feeding relative to illumination alone, it shouldn't be assumed that these measurements are representative of this rapidly-exchanging pool under the steady-state growth conditions.
Thank you very much for raising this important point. We are very much aware of the difficulties to determine the redox state of NADH:NAD+ using these kits. However, there is no other method available that properly distinguishes NADH and NADPH. Furthermore, the centrifugation step was done at -9°C which should minimize metabolic reactions during this step. However, we now added in vivo measurements using the NAD(P)H-module available for the PAM and using the Dual-KLAS/NIR to determine the redox state of ferredoxin (newly added Fig. S4). Both methods show that NAD(P)H as well as ferredoxin are more strongly reduced under photomixotrphic conditions in comparison to photoautotrophic conditions and thus support our previous data.
Reviewer #2 (Public Review):
The observation that cyanobacteria can use two alternative pyruvate decarboxylating enzymes using either NAD+ or ferredoxin is an interesting and the work is useful contribution. The authors very nicely characterize the enzymatic properties of the two pyruvate metabolizing enzymes and also are able to connect the ideas of redox balance with a set of ferredoxins. Even though they are not able to definitively characterized the specific ferredoxin which interacts with the enzyme, the analysis is nicely conducted and it's clear that the suggestion they're making regarding the involvement of the minor ferredoxins is compelling. However, the work could be written in a way that might be more useful.
Specific comments:
Overall this is an interesting study, but the arguments could be sharpened and better connected with the literature. The introduction needs to be considerably revised in my opinion. It is not obvious whether it is even appropriate to discuss the enzymes as an aerobic enzymes or aerobic enzymes, since this concept is simplistic and perhaps, archaic. Indeed, placing the results of the present study in the context of "aerobic enzymes versus aerobic enzymes" is a bit of a 'strawman' argument. For example, the counter examples of O2-tolerant enzymes cited seem to suggest that PFORs have been capable of evolving into O2-tolerant enzymes quite readily and that two types of decarboxylase have evolved for quite different reasons than simple replacement for a new environment. Instead, I think a more current and general perspective relates more to the interpretation that the authors are already putting forth. Namely, the enzymes are utilized according to redox balance considerations rather than sensitivity to oxygen.
Therefore, I think the very long and pedantic introduction is useful for review, but only if it is shortened and also includes the alternative interpretation regarding adaptations to redox potential in the cytoplasm. My guess is that there are plenty of examples of redox balance function arguments in the literature to refer to in contrast to the evolutionary replacement argument used. Certainly, there are good examples regarding glucose toxicity in mutants of Synechocystis that can be considered.
Thank you very much for this point. The O2-tolerant PFORs mentioned were merely shown to be stable in the presence of oxygen in vitro which means that they can be isolated under anaerobic conditions. However, all enzymatic in vitro assays required anaerobic conditions. Only one PFOR was shown to be active in the presence of oxygen in vitro. Physiological studies on the importance of these enzymes under aerobic conditions in vivo are completely missing. However, animated by the requests of the reviewers we searched the literature intensively again and indeed found a recent report, which describes the involvement of PFOR in redox regulation in an aerobic culture of an E. coli mutant, in which glucose-6P dehydrogenase (ZWF) was down-regulated (1). We included this study both in our introduction and discussion. It very much supports our own findings, as the E. coli PFOR requires likewise anoxic conditions in in vitro enzyme tests. We agree that the idea that PDH complex and PFOR are exclusively regulated by oxygen availability might sound simplistic. However, we do not fully agree that this is a strawman argument as both enzyme systems are still mostly discussed as counterparts for either aerobic respiration (PDH complex) or anaerobic fermentation (PFOR)(4). To the best of our knowledge, the study that was included now and our own data, are the very first ones that put clearly forward the idea, that redox control governs the activity of these enzyme systems at the pyruvate node independent of oxygen. However, doubts about the rather simplistic distinction between aerobic versus anaerobic enzymes in general have indeed been expressed. Even though these studies in general lack physiological in vivo experiments. We therefore included this information in the introduction as well. (line 76: There are several reports on the aerobic expression of enzymes that are assigned to anaerobic metabolism in prokaryotes and eukaryotes and therefore challenge the simplistic distinction between aerobic versus anaerobic enzymes (5-7). Their physiological significance and regulation are only partly understood.) This did not result in a shortened introduction though as additional information was added. The new introduction thus includes alternative interpretations as requested and is therefore hopefully more balanced.
Given the interpretation that the alternative forms of the enzyme help cells adjust their redox balance to different conditions, such as photomixotrophic growth, the very nice enzymatic analysis and growth studies of the mutants work would be significantly strengthened by more direct physiological measurements that report intracellular redox states.
Thank you very much for this important point. Intracellular redox states were shown by measurements of the NAD+/NADP level (Figure 1B) and were now extended by new in vivo measurements that show that both the NAD(P)H and the ferredoxin pools are more reduced under photomixotrophic in contrast to photoautotrophic conditions (new Fig. S4).
Minor comments:
line 211: Perhaps, "..the deleted alleles failed to segregate, keeping some wild type copies."
This was changed to: the deleted alleles of fx2 (sll1382) and fx5 (slr0148) failed to segregate, keeping some wild type copies.
It would be interesting to characterize whether the observed distribution of PFOR correlates with specific physiological features. In other words, PFOR seems to become important upon the addition of an external carbon source in way that must integrate with autotrophic metabolism (i.e. mixotrophic growth) altering the balance of the oxidized and reduced form of redox cofactors--does the observed distribution correlate at least with the metabolic characteristics of the handful that have been studied in the lab?
Thank you very much for this suggestion. We checked the lists of cyanobacteria that either possess or do not possess a PFOR in order to search for shared known physiological features. However, the challenge is currently that the number of uncharacterized cyanobacteria in our list is too large. It is therefore impossible to find solid correlations. But we fully agree that it would be interesting to find these.
A more detailed set of calculations that help explain panel C in figure 4 need to be included to support the quoted values for redox potential in free energy. I assume these are standard values and and the specific superscripts and subscription associate with the ΔG nomenclature needs to be defined.
The calculations are shown in the materials and methods part. A respective notice (for calculations see materials and methods part) is now given in the legend of Fig. 4C. Information concerning the nomenclature is found in the cited literature in the materials and methods part as well.
Reviewer #3 (Public Review):
The manuscript by Wang et al. conclusively demonstrates that the cyanobacterium Synechocystis sp. PCC6803 prefers to use the ferredoxin-reducing enzyme PFOR over the NAD+-reducing PDH-pathway when grown under photomixotrophic conditions while the PDH-route is favored under photoautotrophic conditions. Both the potential physiological meaning of this switch and implications for the evolutionary history of the role of the respective enzymes and their pathways are discussed.
The main hypothesis of this work considers that PFOR-mediated decarboxylation of pyruvate replaces the PDH-based one when cells shift from photoautotrophic to photomixotrophic growth conditions. This hypothesis is assessed via the comparison of growth curves measured on a host of deletion mutants and via direct detection of expression levels of certain enzymes. The authors' hypothesis is robustly supported by the majority of the reported experiments and the reviewer is fully convinced by these data. However, I would hold that the data shown with respect to phosphorylation of PDH (Fig. S4) are unconvincing. I can't see a clear difference in growth-curves for the incriminated mutants deltaspkB and L which would convincingly exceed the variation observed for the entire dataset.
We agree that the data on the phosphorylation of the PDH complex including the kinase mutants are not very convincing. We were uncertain from the beginning on whether it would be a good idea to include these data sets and therefore discussed them very cautiously in the manuscript. Anyway, as the enzymatic tests with the E3 subunit of the PDH complex at different NADH concentrations show convincingly that high NADH levels have an inhibitory effect on the complex, we now decided to delete both data sets out of the manuscript, as they are not really required for the statement of the manuscript.
S. Li et al., Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli. Metab Eng 64, 26-40 (2021).
T. Nakayama, S. Yonekura, S. Yonei, Q. M. Zhang-Akiyama, Escherichia coli pyruvate:flavodoxin oxidoreductase, YdbK - regulation of expression and biological roles in protection against oxidative stress. Genes Genet Syst 88, 175-188 (2013).
A. Witt, R. Pozzi, S. Diesch, O. Hädicke, H. Grammel, New light on ancient enzymes – in vitro CO2 Fixation by Pyruvate Synthase of Desulfovibrio africanus and Sulfolobus acidocaldarius. The FEBS Journal 286, 4494-4508 (2019).
M. Müller et al., Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes. Microbiology and Molecular Biology Reviews 76, 444 (2012).
S. B. Gould et al., Adaptation to life on land at high O2 via transition from ferredoxin-to NADH-dependent redox balance. Proceedings of the Royal Society B: Biological Sciences 286, 20191491 (2019).
O. Schmitz, J. Gurke, H. Bothe, Molecular evidence for the aerobic expression of nifJ, encoding pyruvate : ferredoxin oxidoreductase, in cyanobacteria. FEMS Microbiol. Lett. 195, 97-102 (2001).
K. Gutekunst et al., LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator. Molecular Microbiology 58, 810-823 (2005).
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Evaluation Summary:
The work supports the hypothesis that novel specific enymes evolved to modify metabolic pathways, allowing phototrophs to shift from growth under photoautotrophic and photomixotrophic growth conditions.
(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. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In Wang et al., the authors investigate issues related to the relative proportion of flux for the enzymatic decarboxylation of pyruvate between PDH (pyruvate dehydrogenase) and PFOR (pyruvate-ferredoxin oxoreductase) in the model organism Synechococystis. The manuscript provides evidence that PDH becomes increasingly inactivated by a high ratio of NADH:NAD+ as well as evidence to suggest that PFOR is transcribed and remains intact under aerobic conditions. The authors put forward the theory that both PDH and PFOR are functionally active routes for pyruvate decarboxylation under aerobic conditions, whereas PFOR has previously been assumed to be inactive under growth conditions containing oxygen. This distinction is particularly highlighted by conditions where Synechocystis is grown photomixotrophically - and …
Reviewer #1 (Public Review):
In Wang et al., the authors investigate issues related to the relative proportion of flux for the enzymatic decarboxylation of pyruvate between PDH (pyruvate dehydrogenase) and PFOR (pyruvate-ferredoxin oxoreductase) in the model organism Synechococystis. The manuscript provides evidence that PDH becomes increasingly inactivated by a high ratio of NADH:NAD+ as well as evidence to suggest that PFOR is transcribed and remains intact under aerobic conditions. The authors put forward the theory that both PDH and PFOR are functionally active routes for pyruvate decarboxylation under aerobic conditions, whereas PFOR has previously been assumed to be inactive under growth conditions containing oxygen. This distinction is particularly highlighted by conditions where Synechocystis is grown photomixotrophically - and where the NADH:NAD+ pool may be relatively over-reduced because of two parallel inputs of reductant (water-splitting at PSII and catabolism of glucose). The authors examine growth under photoautotrophic and photomixotrophic conditions for a number of relevant mutants including members of the ferredoxin/flavodoxin family, PFOR, and NDH-1 complex subunits.
The theory put forward in this manuscript is of general interest regarding electron flux through the combined electron transport chain (photosynthetic + respiratory) of cyanobacteria. The authors further broaden the potential audience for the manuscript by elaborating on the potential significance of these results in the context of a switch from PFOR (ancestral) to PDH (oxygenic/modern).
Comments:
Generally, theories put forward in this manuscript are intriguing and have a number of potential implications for understanding electron flux and regulation of central metabolic processes in photosynthetic microorganisms. If these theories are supported and become more generally adopted, they would have significant impact on the understanding of the regulation of central carbon metabolism in cyanobacteria. That said (due in no small part to the complexity of some of these pathways), the evidence provided to support the hypotheses is indirect in many instances. In some cases, there is a pairing of indirect data with broad statements that can come across as over-reach. These problems can be somewhat exacerbated by an unclear organization at parts of the Discussion, a lack of succinctly defined claims, and numerous typographical considerations.Major considerations:
A major component of the proposed theories in this manuscript rest upon the assumption that PFOR is an active enzyme under highly aerobic conditions: this claim is never directly demonstrated. Indirect evidence of altered growth of pfor mutants, increased repression of PDH, and the higher NADH:NAD+ ratio under photomixotrophic conditions is in general alignment with this theory. However, while deletion of pfor does indeed result in altered growth dynamics in Synechocystis under periods of photomixotrophy, the alterations do not entirely align with the idea that this pathway is critical for rapid growth under aerobic conditions. For instance, pfor and most of the highlighted mutants (fdx 3, fdx 9, isiB) presented in Figure 3 show the greatest defects in their OD after reaching stationary phase (more rapid decline in OD on/after Day 6) relative to WT. This doesn't align as nicely with the highest NADH:NAD+ seen in Days 3-5 (which is also specifically called out: e.g., Line 146, Supplemental Figure S8). In this context, the deletion of F-GOGAT is much more convincing in it's severity and timing, yet for this mutation to have a more severe phenotype is unexpected if PFOR is one of the primary/sole electron donors to the ferrodoxin pool from glucose utilization as proposed (i.e., stated differently, F-GOGAT is only one of the enzymes downstream of ferrodoxin and might be expected to have a more subtle phenotype in comparison to the KO of PFOR if that is a primary source for electrons to ferredoxin under photoheterotrophic conditions).A central tenant of the argument put forward on the evolutionary importance of using either PFOR vs. PDH is the conservation of extra free energy by the former reaction. However, additional information on the ferredoxin paralog(s) that accept electrons from PFOR is necessary to evaluate these claims. Based on the data within these manuscripts, Fdx3, Fdx9, and IsiB have the strongest links to PFOR: though the authors do take care to never state directly that they have evidence that these are the acceptors in vivo. Given the variability in the midpoint potentials of different ferredoxins, some ferredoxin acceptors may better conserve the free energy in pyruvate, while others may actually be more 'wasteful' than NAD+ as the acceptor through PDH. Unfortunately, the midpoint potentials for Fdx3, Fdx9, and IsiB are unknown or not stated in this manuscript. It is therefore unclear what ferredoxin is being used as the reference point for conservation of Gibbs free energy in Figure 4C and referenced multiple times in the text.
Finally, the measurements of NADH:NAD+ (most prominently used for measurements in Fig 1B) utilized kits that require multiple, long centrifugation steps in the dark prior to assaying this rapidly exchanging pool. While it appears that the authors were able to get reproducible results with these kits, it is difficult to interpret what the increase in relative NADH levels in glucose-fed cells means given that 10+ minutes of incubation in the dark and/or changing temperatures elapsed after the cyanobacteria were removed from the incubator before the NADH:NAD ratio was assessed. While it superficially makes logical sense that the cytosol would be over-reduced when illuminated and under glucose feeding relative to illumination alone, it shouldn't be assumed that these measurements are representative of this rapidly-exchanging pool under the steady-state growth conditions.
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Reviewer #2 (Public Review):
The observation that cyanobacteria can use two alternative pyruvate decarboxylating enzymes using either NAD+ or ferredoxin is an interesting and the work is useful contribution. The authors very nicely characterize the enzymatic properties of the two pyruvate metabolizing enzymes and also are able to connect the ideas of redox balance with a set of ferredoxins. Even though they are not able to definitively characterized the specific ferredoxin which interacts with the enzyme, the analysis is nicely conducted and it's clear that the suggestion they're making regarding the involvement of the minor ferredoxins is compelling. However, the work could be written in a way that might be more useful.
Specific comments:
Overall this is an interesting study, but the arguments could be sharpened and better connected …
Reviewer #2 (Public Review):
The observation that cyanobacteria can use two alternative pyruvate decarboxylating enzymes using either NAD+ or ferredoxin is an interesting and the work is useful contribution. The authors very nicely characterize the enzymatic properties of the two pyruvate metabolizing enzymes and also are able to connect the ideas of redox balance with a set of ferredoxins. Even though they are not able to definitively characterized the specific ferredoxin which interacts with the enzyme, the analysis is nicely conducted and it's clear that the suggestion they're making regarding the involvement of the minor ferredoxins is compelling. However, the work could be written in a way that might be more useful.
Specific comments:
Overall this is an interesting study, but the arguments could be sharpened and better connected with the literature. The introduction needs to be considerably revised in my opinion. It is not obvious whether it is even appropriate to discuss the enzymes as an aerobic enzymes or aerobic enzymes, since this concept is simplistic and perhaps, archaic. Indeed, placing the results of the present study in the context of "aerobic enzymes versus aerobic enzymes" is a bit of a 'strawman' argument. For example, the counter examples of O2-tolerant enzymes cited seem to suggest that PFORs have been capable of evolving into O2-tolerant enzymes quite readily and that two types of decarboxylase have evolved for quite different reasons than simple replacement for a new environment. Instead, I think a more current and general perspective relates more to the interpretation that the authors are already putting forth. Namely, the enzymes are utilized according to redox balance considerations rather than sensitivity to oxygen. Therefore I think the very long and pedantic introduction is useful for review, but only if it is shortened and also includes the alternative interpretation regarding adaptations to redox potential in the cytoplasm. My guess is that there are plenty of examples of redox balance function arguments in the literature to refer to in contrast to the evolutionary replacement argument used. Certainly there are good examples regarding glucose toxicity in mutants of Synechocystis that can be considered.
Given the interpretation that the alternative forms of the enzyme help cells adjust their redox balance to different conditions, such as photomixotrophic growth, the very nice enzymatic analysis and growth studies of the mutants work would be significantly strengthened by more direct physiological measurements that report intracellular redox states.
Minor comments:
line 211: Perhaps, "..the deleted alleles failed to segregate, keeping some wild type copies."It would be interesting to characterize whether the observed distribution of PFOR correlates with specific physiological features. In other words, PFOR seems to become important upon the addition of an external carbon source in way that must integrate with autotrophic metabolism (i.e. mixotrophic growth) altering the balance of the oxidized and reduced form of redox cofactors--does the observed distribution correlate at least with the the metabolic characteristics of the handful that have been studied in the lab?
A more detailed set of calculations that help explain panel C in figure 4 need to be included to support the quoted values for redox potential in free energy. I assume these are standard values and and the specific superscripts and subscription associate with the ΔG nomenclature needs to be defined.
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Reviewer #3 (Public Review):
The manuscript by Wang et al. conclusively demonstrates that the cyanobacterium Synechocystis sp. PCC6803 prefers to use the ferredoxin-reducing enzyme PFOR over the NAD+-reducing PDH-pathway when grown under photomixotrophic conditions while the PDH-route is favored under photoautotrophic conditions. Both the potential physiological meaning of this switch and implications for the evolutionary history of the role of the respective enzymes and their pathways are discussed.
The main hypothesis of this work considers that PFOR-mediated decarboxylation of pyruvate replaces the PDH-based one when cells shift from photoautotrophic to photomixotrophic growth conditions. This hypothesis is assessed via the comparison of growth curves measured on a host of deletion mutants and via direct detection of expression …
Reviewer #3 (Public Review):
The manuscript by Wang et al. conclusively demonstrates that the cyanobacterium Synechocystis sp. PCC6803 prefers to use the ferredoxin-reducing enzyme PFOR over the NAD+-reducing PDH-pathway when grown under photomixotrophic conditions while the PDH-route is favored under photoautotrophic conditions. Both the potential physiological meaning of this switch and implications for the evolutionary history of the role of the respective enzymes and their pathways are discussed.
The main hypothesis of this work considers that PFOR-mediated decarboxylation of pyruvate replaces the PDH-based one when cells shift from photoautotrophic to photomixotrophic growth conditions. This hypothesis is assessed via the comparison of growth curves measured on a host of deletion mutants and via direct detection of expression levels of certain enzymes. The authors' hypothesis is robustly supported by the majority of the reported experiments and the reviewer is fully convinced by these data. However, I would hold that the data shown with respect to phosphorylation of PDH (Fig. S4) are unconvincing. I can't see a clear difference in growth-curves for the incriminated mutants deltaspkB and L which would convincingly exceed the variation observed for the entire dataset.
While I consider the results part as describing (largely) well-performed and pertinent experiments, I have a few issues with the discussion and conclusion sections. To my mind these sections contain a few unwarranted conclusions and misconceptions which need straightening out prior to publication.
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