Nitrogen signaling factor triggers a respiration-like gene expression program

  1. Shin Ohsawa
  2. Michaela Schwaiger
  3. Vytautas Iesmantavicius
  4. Rio Hashimoto
  5. Hiromitsu Moriyama
  6. Hiroaki Matoba
  7. Go Hirai
  8. Mikiko Sodeoka
  9. Atsushi Hashimoto
  10. Akihisa Matsuyama
  11. Minoru Yoshida
  12. Yoko Yashiroda
  13. Marc Bühler

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Abstract

Microbes have evolved intricate communication systems that enable individual cells of a population to send and receive signals in response to changes in their immediate environment. In the fission yeast Schizosaccharomyces pombe , the oxylipin Nitrogen Signaling Factor (NSF) is part of such communication system, which functions to regulate the usage of different nitrogen sources. Yet, the pathways and mechanisms by which NSF acts are poorly understood. Here, we show that NSF physically interacts with the mitochondrial sulfide:quinone oxidoreductase Hmt2 and that it prompts a change from a fermentation- to a respiration-like gene expression program independently of the carbon source. Our results suggest that NSF activity is not restricted to nitrogen metabolism alone and that it could function as a rheostat to prepare a population of S. pombe cells for an imminent shortage of their preferred nutrients.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript the authors are interested in understanding how fission yeast respond to a Nitrogen Signaling Factor (NSF) that has previously been shown to allow Leucine auxotrophs to grow in the presence of Leucine when Nitrogen Catabolite Repression (NCR) is triggered by the presence of a high quality Nitrogen source such as Ammonium Chloride (NH4Cl).

    The authors begin with a screen to identify genes that affect the ability of wild type cells grown near cells with leucine auxotrophy to enhance or abolish NCR phenotype. They screened the non-essential gene deletion library which they manipulate so that it only contains a leucine auxotrophy (unlike the original gene deletion library which contains additional auxotrophies). They identify 137 genes whose deletion allows growth of Leu auxotrophs in the presence of Leucine and Ammonia without the presence of WT cells. These genes are required for NCR. They further identify 203 genes which do not bypass NCR even in the presence of wild type cells, and are thus important for bypassing NCR in the presence of WT cells.

    They then conduct a second screen to identify which of these genes are important for bypassing NCR in response to the Synthetic NSF, 10(R)-hydroxy-8(Z)-octadecenoic acid, by looking for genes which grow in the presence of leucine when ammonia is not present, but do not grow in the presence of leucine when ammonia is present, even when NSF is added. This second screen identifies 117 strains carrying deletions in a gene set enriched for genes related to cellular respiration and mitochondria. They then show that the NSF bypass of NCR is linked to respiration by showing that it is abolished in the presence of the respiration inhibitor Antimycin A, that growth in low levels of glucose can bypass NCR in the absence of NSF< and that cells supplemented with NSF have a higher oxygen consumption rate.

    To gain insight into how the cell responds to NSF, the authors then gather RNA expression data from cells grown in high ammonium concentrations following treatment with NSF relative to a negative control treated only with Methanol (the vehicle into which NSF is dissolved). They argue that the gene expression pattern resembles gene expression data from cells undergoing respiration in glycerol relative to cells undergoing fermentation in glucose. They show that the upregulated genes relate to trehalose synthesis, detoxification of Reactive Oxygen Species, and cellular fusion and the downregulated genes are related to cellular adhesion and flocculation.

    They validate their RNA-seq measurements by showing that the two most highly induced and two most highly repressed genes respond to NSF addition in a dose dependent manner and do not respond oleic acid which is chemically similar to NSF. The most highly responsive gene they identify is an uncharacterized gene, SPBPB2B2.01, which they suggest naming "NSF-responsive amino acid transporter 1" (nrt1). They also show that the nrt1 response is dependent on the culture density, and that the response is present (though the magnitude varies) in YES and in EMM under varying nitrogen concentrations, and that yfp driven by the nrt1 promoter is induced by NSF.

    The authors then investigate the 8 transcription factors that were present in their list of genes required for NSF-mediated adapted growth. They note that Hsr1 was the only one of these transcription factors, indeed the only gene, that was a hit in their screen for NSF-mediated adapted growth and whose expression was induced upon NSF treatment. To see if the activity of the other transcription factors changed in response to NSF treatment, the authors then gathered ChIP-seq data using 6 of these transcription factors as targets for IP. They saw that for Hsr1 and Php3, targets that had increased RNA-seq expression showed an increase in promoter occupancy while for Hsr1, Php3, Adn2, and Atf1, genes that had decreased RNA-seq expression showed a decrease in promoter activity.

    Finally the authors attempt to identify the mode of action of NSF by generating a functionalized NSF with an alkyne tag (AlkNSF) which they then use as a probe to identify NSF binding partners. They first show that AlkNSF does allow bypass of NCR, although at 30-fold higher concentration. Also AlkNSF induces nrt1 expression in a dose dependent manner, although the expression saturates at a lower level and requires a much higher concentration for induction. They then look for proteins that co-purify with AlkNSF compared to a control that was pre-incubated with NSF which was expected to compete off AlkNSF. The only significant protein they saw was Ayr1, which was not identified in their screen and which did not abrogate NSF bypass of NCR when deleted independantly. They saw that Ayr1 deletion actually increases the response of nrt1 and mei2 targets to NSF, and speculate that Ayr1 metabolises NSF and reduces the cell's ability to respond to NSF to bypass NCR.

    They then repeat the affinity purification / mass spec protocol in an Ayr1 delete cells to identify other interaction partners, this time incubating with a higher concentration of NSF, and also comparing to an experiment using Alkeyne Oleic Acid as a control for non-specific binding. The top two specific hits from this assay are Hmt2 and Gst3. NSF was still able to rescue NCR in gst3 deletes, indicating that it was not relevant for the phenotype. Cells lacking hmt2 did not grow in EMM, but did grow in YES when not supplemented with ammonium and when supplemented with ammonium did not grow, and addition of NSF did not rescue growth. They also see that nrt1 and mei2 gene induction in response to NSF is abolished when hmt2 is deleted. They then argue that hmt2, a sulfide:quinone oxidoreductase localized in the inner membrane of mitochondria is a direct target of NSF that triggers a switch to respiratory metabolism and allows bypass of NCR.

    Below are comments that I think ought to be addressed prior to publication (Major comments)

    1. In line 70, the authors state that "S. pombe cells rely on their own BCAA synthesis to sustain growth" when grown alongside Leucine when ammonium is supplied in the media. If prototrophs can inhibit NCR via NSFs in neighboring auxotrophic cells on the same plate, couldn't they also inhibit NCR within their own colony? How do we know that prototrophic cells grown in high quality nitrogen sources along with, say leucine, are not taking up leucine? The fact that leucine auxotrophs cannot grow in high quality nitrogen sources when leucine is present does not imply that wild type cells must use be synthesizing BCAAs rather than importing them. In a recent paper (Kamrad et al Nat. Microbiol. 2023, https://www.nature.com/articles/s41564-022-01304-8), it was shown that S. cerevisiae cells grown in lysine and in high concentrations of ammonium uptake lysine rather than synthesize it as lysine concentrations in the media are increased. I am aware via unpublished results that this is the case for Leucine as well. I would be surprised if the same isn't true in S. pombe. The authors should caveat or remove this assertion.
    2. It is important for the authors to put their observation linking respiration to rescue from NCR in context with findings from a closely related study (Chiu et al 2022) which included some authors from this manuscript and which the authors cite. In that paper, it was shown that the siderefore ferrichrome can also rescue NCR in fission yeast. That paper stated "It is likely that ferrichrome increased mitochondrial activity, which enabled efficient utilization of glucose downstream of the glycolytic pathway" based on experiments in different concentrations of glucose. This evidence seems to support the link between respiration and rescue from NCR proposed by the authors of this manuscript. The authors should acknowledge this closely related and earlier work as it strengthen's the case they are trying to make. They could even test if ferrichrome addition makes cells sensitive to antimycin A (as in fig 1E), but that extra experiment would be optional in my opinion.
    3. In figure 1B for the second screen I do not understand what the photos represent. For the photos, two rows are meant to have no NH4 and also no NSF and the label on that image makes no mention of Leucine supplementation. In the diagram there are two rows that have NH4 and leucine and one row that has no NH4 but does have leucine. I assume the diagram is correct and the labels on the images are incorrect.
    4. It would be important for the authors to put their observation linking respiration to rescue from NCR in context with findings from Chiu et al 2022 which the authors cite. In that paper, it was shown that the siderefore Ferrichrome can also rescue NCR in fission yeast which the authors site which found that a siderephore rescues NCR. Also the authors of that paper stated "It is likely that ferrichrome increased mitochondrial activity, which enabled efficient utilization of glucose downstream of the glycolytic pathway." based on experiments in different concentrations of glucose. This evidence seems to support the link between respiration and rescue from NCR proposed by the authors of this manuscript.
    5. In line 133. The authors state that the 29 mutants that didn't grow under Leucine supplementation either without NH4CL or with NH4Cl whether or not NSF was present were "related to EMM Growth, leucine uptake, or utilization of ammonium as the sole nitrogen source." The first two make sense, but I can't see why a a strain with deletion of a gene related to utilization of ammonium as a sole nitrogen source wouldn't grow when supplemented with leucine. In fact for all the leucine auxotrophs in the screen, if one was to try to grow them with ammonium as the sole nitrogen source they would not grow, so it isn't clear that this screen can identify genes responsible for utilization of ammonium as a sole nitrogen source. The authors should clarify or remove this point.
    6. 203 strains are important for avoidance of NCR (because in the presence of Ammonium and Leucine, as well as a WT strain, they cannot grow). Of these 57 strains can't grow in the presence of a WT strain but they can grow in the presence of NSF. The authors conclude in line 138 that these strains are "likely to respond to a transmissible signal that is different from NSF". This is confusing because deletion of these genes still does allow cells to respond to NSF, however when these cells are growing in the presence of wild type cells (which in their model are releasing NSF), the cells don't grow. I am confused about the nature of the transmissible signal that the authors suggest. It would appear that when these genes are deleted and grown next to a wild type cell which sends the alternative signal and the NSF, the other transmissible signal would inhibits the ability of NSF to release NCR (as NSF can still rescue the gene). It is not clear how the other transmissible signal would work when the gene is present as it is clearly not necessary to rescue growth.

    A simpler explanation might be that there was contamination in the second screen, or that there was a threshold effect - perhaps in the first screen the strains grew just below a threshold and in the second screen it grew just above that level.

    The authors should clarify their interpretation for these strains, and acknowledge any alternative technical explanations.

    1. The authors' efforts to removed confounding effects that might stem from additional auxotrophic alleles made the screen more convincing. However, Fig 1E, 1F, 5B, and 5E were done with EMM+Leu+Ade+Ura, while the initial strain was just done in the presence of additional Leucine. It is unclear why this was done from the text and captions, but I assume it was because they used a strain that was ade- and ura- in addition to being leu-. Given that they had strains without these additional mutations, this seems like a strange choice. The authors should acknowledge that there are possible confounding effects of adding adenine and uracil to the media, and, if they did have additional metabolic deletions, acknowledge that that could possibly be confounding.
    2. Fig 1E, it appears that cells can grow without NSF in the presence of ammonium and additional amino acids after 10 days (although NSF is required for growth at 5 days). This is not a problem for the screen as that was taken at 5-6 days, but it appears as though NSF does not rescue growth so much as speed it up. The authors should acknowledge this when describing the phenotype. It also argues for a quantitative time course growth experiment to compare growth over the course of 10 days with and without NSF, although this would not be necessary to the paper's main argument.
    3. In line 191 and 192, the authors suggest that the "downregulation of flocculation/adhesion related genes by NSF could serve to avoid undesirable mating during growth". If this is the case, I don't understand why mating genes and cellular fusion genes would be upregulated. What do the authors mean by undesirable mating? Wouldn't flocculation increase desirable mating as well? If all mating is undesirable, wouldn't upregulation of mating and cellular fusion genes be detrimental?
    4. The authors mention that trehalose is an antioxidant, for which they reference Malecki 2019, however that paper shows no direct evidence of trehalose functioning as an antioxidant under respiratory conditions. It only shows that some trehalose synthesis genes are upregulated when cells are grown under glucose. The authors should identify primary literature to back this statement up, or soften the wording. Also trehalose is known to be a storage metabolite (which is mentioned in Malicki et al 2019, but not in this manuscript). In fact work in budding yeast has show that trehalose can be a shared metabolite that can be produced by respiring cells and used as a fermentable carbon source in communities of budding yeast cells that consist of fermenting and non-fermenting cells (Varahan et al, eLife 2019 https://doi.org/10.7554/eLife.46735). It seems that this role should be considered as an alternative explanation for the induction of trehalose in respiratory cells.
    5. Line 208: The stimulatory effect of NSF on NRT1 decreased with cell density, thus cell density is likely to be an important factor in terms of gene expression. The methods section, text and figure captions do not mention the density at which cells were inoculated/harvested for RNA-seq and other experiments. If that density was more than OD 0.1, then this would be inconsistent with the measurements from Fig 3. Also in fig 3D, The culture density is not mentioned in the figure or the caption, even though the text suggests that for that experiment cells were grown at low density (Lines 212-213). The authors should provide information on density for their experiments in order for them to be reproducible, as they show it is a key factor.
    6. In suggesting a name for NRT1 (NSF-responsive amino acid transporter 1), the authors assume that the gene has a role in amino acid transmembrane transport, but they have no experiments showing this phenotype. They mention that it is Inferred from homology with other amino acid transporters. I presume this name has already been approved by Pombase and is not provisional, but it seems that including phenotypes inferred from homology, rather than from experiments is unwise. Do the authors have any other direct evidence that this is a bona fide Amino Acid Transporter? Perhaps a name like "NSF-responsive gene" would be more appropriate.

    Related to this, it appears that the expression level of Nrt1 may be very low (see Fig S2B in which the scale of the RNA-seq track is very small [-1,1] and the amount of expression is very small even when NSF is added). Looking at Fig 2A, the total transcript abundance did not appear to be very low in terms of counts per million (over 100) is this a discrepancy in fig S2B? Perhaps the large fold change is the result of counts very close to zero in the control condition? Also in Fig 3 the nrt1 expression levels did not appear to be especially low and they appeared repeatable. Is the RNA-seq data shown in fig S2B for nrt1 a fluke or am I misinterpreting it?

    1. To show that their Chip-seq worked, the authors showed specific examples of Chip-seq reads for target genes Line 240, "Previously determined target genes of these TFs were significantly enriched in our data set, demonstrating that the experiment has worked (Figure S2A)." Is the significance here, the threshold from fig S2B? If so that threshold should be clearly stated here in the text. If it is the fact that asn1 shows up as "Fil1 bound" is strange as there are no genes that had significant changes in ChIP-seq signals for fig S2B. If there is another threshold the authors should describe it. While some of the examples they showed were convincing (e.g. php3-flag for the php3 regulated gene gln1 and the increased reads for srw1 for the reb1 target srw1), there were some targets that didn't seem to be especially enriched for their designated transcription factor. For example, the gene trx1 which was identified as an Hsr1 binding target had some binding from Hsr1, but more from Php3 and equivalent amounts for many of the other transcription factors. A clear description of how genes are chosen to be significant in the text, alongside references/selection criteria the authors used to select the specific genes shown should be provided to improve reproducability.
    2. In lines 244-246 the authors state that "These differences in TF occupancy were positively correlated with target gene expression changes. That is, individual genes that were upregulated by NSF tended to be more strongly bound by the TFs, whereas downregulated genes were less occupied by the respective TFs (Figure 4A)." This is far from a general trend. The trend is not there for reb1 and fil1. In fact fil1 looks to the eye like it shows a decrease in occupancy for genes with increased expression, and I worry that the authors did a one sided test for significance that would have missed this, although the variability of the genes that don't change in this case is very high, so there could be no significant effect. The authors elaborate on some of the detail in following statements, but they should soften or remove this statement.

    Related to this, in line 254, the authors state: "These results imply that NSF exposure rewires the recipient cell's transcriptional program, for which the TFs Atf1, Adn2, Adn3, Fil1, Hsr1, Php3, Php5, and Reb1 are indispensable (Table S3)." While I am convinced from the RNA-seq evidence and some of the chip-seq evidence that NSF exposure rewires cell's transcriptional program, I am not convinced that the 8 transcription factors they mention are indespensable for rewiring the transcriptional program. While they may be indespensible for the phenotype itself, Reb1, and Fil1 show no no siginificant enrichment in occupancy of upregulated or downregulated targets (Fig 4A) and, along with Atf1, Reb1, and Fil1, have very few genes in which ocupancy is changed significantly (Fig S2B), while no chip-seq experiments were shown for Php5 and Adn3.

    The more specific summary of the data (Lines 250-253) from Fig S2B describing how hsr1 and adn2 have the strongest effects of the transcription factors required for NSF-mediated NCR bypass is a much stronger message for this section.

    1. In line 335, the authors state that "in contrast to other communication systems, NSF does not induce noticeable changes in S. pombe's morphology", referring to changins in mating, filamentation, and bacterial biofilm formation. However they do show very clearly that NSF does cause a large decrease in expression in flocculation/adhesion genes. The fact that they do not see a change in morphology is likely due to the fact that the lab strain in the conditions used for this assay do not flocculate. We have recently identified conditions and strains which do exhibit flocculation in this preprint [https://www.biorxiv.org/content/10.1101/2023.12.15.571870v2]. It is likely that if they had a strain and conditions that did flocculate addition of NSF would break up flocculation and thus change the morphology based on their evidence. The authors should remove or caveat this point.
    2. Line 270 Fig 5B: The concentration of NH4Cl listed in the text (374mM) does not match the concentration shown on the figure (748mM). I assume this is a typo but it should be corrected prior to publication.

    Also I have several minor comments to help improve the manuscript.

    m1: Lines 66-70- state that "uptake of the branched-chain amino acids (BCAA) isoleucine (Ile), leucine (Leu), and valine (Val) is suppressed in the presence of high-quality nitrogen sources such as ammonium or glutamate, because the expression of transporters or permeases that are needed for the uptake of poorer nitrogen sources are down regulated (Zhang et al, 2018)." This reference is for S. cerevisiae and is a review. The authors should cite original results in S. pombe if possible, and if that is not available, alert the reader that this result is from a different species.

    m2: It is unclear from the methods section how the images taken for the screens were analyzed. Were they analayzed and scored by hand, or using custom image analysis software. Either way, when publishing the authors should publish the scores for each deletion mutant in their screen. If there was custom image analysis, the authors should mention in their methods the cutoffs which they used to score growth, and consider plotting the data as a supplement so readers can get a sense of how sensitive the screen was.

    m3: The authors identify 137 mutants that did not require NSF signaling to bypass NCR and claimed these genes were required for NCR. It would be helpful and give more confidence in this screen to demonstrate the extent to which the genes identified in this study overlap with any previous genes required for NCR, and whether there was any GO-term enrichment in this set.

    m4: It would be interesting if the authors could speculate a bit in their discussion on why mitochondrial respiration counteracts NCR. Is there something about cells undergoing respiration that would make it easier for them to use BCAAs than to produce them, or conversely something about fermenting cells that makes it easier for them to produce BCAAs rather than importing them?

    m5: It is unclear why Figure 1F has 'MP biomedicals TM' listed in the figure. It doesn't seem to be listed in the caption or the methods. Is this different media than in other experiments? If so, the authors should add that information to the methods or the caption.

    m6: In Line 160, positively influenced is strange wording, do the authors mean "induced"?

    m7: In the section on gene expression change upon exposure to NSF, the authors use a + after each gene name. My understanding is that that notation is meant to refer to strains with the wild type genotype of that gene, and not the gene itself. Shouldn't the gene be italicised in lower case to represent the gene? See: Lera-Ramirez et al 2023 https://doi.org/10.1093/genetics/iyad143.

    m8: In Fig 2A, genes are displayed on a plot that depicts level vs log2FC, but a comparison between the fold change and p-value would be more useful, and I believe DESeq2 should provide an adjusted p-value for these genes. A related issue is that it appears as though there were no biological replicates, though there was data gathered at different time points. In these genome wide experiments, replicates can give confidence to data and help distinguish true change from intrinsic variability of expression in specific genes. Though the authors did qPCR to validate specific results, it would have improved the quality of their systems-level data to have replicates for these and other key experiments (Chip-seq, affinity purification and even the screen).

    m9: Supp Fig S1: To show that similar gene expression profiles exist for other time points, it would be more convincing to show Log fold change 2h vs 4h and 2h vs 6h and show correlation, or else to make a heat map with all genes to see that genes that go up in one condition go up in the other conditions. It is not clear if the red and blue colors are defined for the 2h dataset and then mapped onto the 4 and 6h dataset, or if they are independently assigned for each plot.

    m10: Mbx2 is a key transcription factor related to flocculation and adhesion genes, and its expression is correlated with expression of its targets. If this transcription factor's expression levels decreased in response to NSF, that might strengthen and help explain the decrease in expression the authors observe in flocculation/adhesion genes when cells encounter NSF. If it it does not change, it might also be interesting for readers interested in these phenotypes.

    m11: In Fig 3D, The notation for the Ammonium concentrations for EMM and YES are inconcistent (+ vs parentheses), also the units (mM from the caption) are not on the figure, but the abbreviation "N" is which is confusing and inconsistent with the other plots in which NH4CL is not abbreviated. Additionally, the caption lists additional nutrients in the media for the EMM conditions (Leu, Ade, Ura) which ought to also be listed.

    m12: In lines 233-235, the authors say "One possibility is that they remain bound to their target genes but become activated or deactivated by NSF directly, or posttranslational modification, such as phosphorylation in the case of Atf1". I don't think the authors intend this, but this sentence could be taken to mean that Atf1 has been shown to be phorphorylated by NSF in the reference they site. I think the authors should clarify, i.e. by saying "..such as phophorylation which is known to regulate activity of Aft1 in response to oxidative and osmotic stress [Lawrence et al 2009]".

    m13: In Fig 4B and Fig S2A, there are grey and colored tracks for the chip-seq (- and + NSF), but they are very difficult to see. If grey is in front it is hard to tell how close the colored peak wehn the colored peak is lower. For example, grey is in front for pex7 while color is in front for yhb1. Could the authors add some transparancy so that the data for both conditions could be seen at once? Also there is little information on the control. My assumption for the input(ChIP) sample was that it was cross-linked and sonicated but not immunoprecipitated, but it is not clear what conditions it was in. I would assume it was done without NSF treatment in WT cells, but those details should be added in the caption or methods. In particular, in the input there is a large spike for Gsf2. Do the authors have any explanation for this and does it have anything to do with that gene's NSF responsiveness?

    m14: The authors might consider putting something like Fig S2B (or even a corresponding volcano plot) as a main figure for Fig 4 in addition to the other two panels, as the individual examples from fig 4B are nice to see, but do not give a broad overview of the data.

    m15: In line 348, the wording "Would score" might be better replaced by "would be identified."

    Significance

    Assessment:

    In general I find the authors arguments compelling and their experiments convincing. The initial and follow on screens were well designed and the authors linked respiration and the action of NSF in a convincing way. The analysis of RNA-seq data was also convincing, especially regarding the decreased expression of flocculation and adhesion genes, and the follow up of specific targets gives confidence in the data (though see Major point 12 below regarding the naming and expression levels of nsf1). The identification of hmt2 as a functional target of NSF was compelling and rigorous, and the authors offer an interesting hypothesis to connect this to respiration that could form the basis of future studies.

    At times I thought that some of the interpretation of the results was hard to follow, poorly worded, or off the mark (see comments below). The presentation of the CHiP seq data also felt incomplete, though the influence of Hsr1 and Adn2 on expression of NSF1 targets was convincing. The genome wide assays (RNA-seq, CHiP seq, screen and pull-down/mass spec) could have done with replicates which would have improved statistics and reliability of the results presented for those experiments, although for key messages, the authors followed up with convincing targeted experiments.

    The study represents an advance on recent work in NCR in fission yeast in linking this with the broad metabolic switch between fermentation and respiration, and in that sense makes this of interest to a broader swathe of the microbiology community, outside those interested in metabolic regulation in microbes. In addition to being of interest to applied researchers interested in producing metabolites with yeast and other microbes, the link to cell signaling and, via flocculation and adhesion genes, to microbial multicellular-like phenotypes would make this work of interest to those interested in microbial communities.

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    Referee #2

    Evidence, reproducibility and clarity

    This paper uses the model system Schizosaccharomyces pombe to investigate how the oxylipin nitrogen signaling factor functions to send signals and adapt the metabolism upon a change in nutrients in the environment. Combining genome-wide screens, RNA-sequencing and chemical biology, the authors find that the nitrogen signaling factor triggers a change from fermentation to a respiratory metabolism, through a direct interaction with a mitochondrial oxidoreductase Hmt2.

    Major comments:

    Overall, the manuscript lacks readability and coherence. Quite a lot of genes/TFs and proteins are mentioned, it is difficult to find a coherent story and clear overview and connection between these subparts. The manuscript would benefit from a general proposed scheme/working mechanism in the discussion and streamlining the results and data into a single biological storyline.

    Several statements or results are not sufficiently clear, elaborated or nuanced. The paper would benefit from more explanation and discussion.

    In the discussion section, the authors are not consistently referencing figures.

    179-181: 'GO enrichment analysis of the 92 downregulated genes' but on line 167, it is '74 downregulated genes' that are mentioned. It is unclear where this difference in number of downregulated genes comes from. Similarly, for the upregulated genes. '156 genes' are mentioned on line 181 but only '98' on line 167.

    189: The statement that the downregulation of flocculation could serve to avoid mating, though sounding logical, is undermined by the finding that mating-related processes are upregulated in the experiment. I find this statement rather speculative

    247-249: The statement is too broad, the effects are visible for maybe 3 TFs, the others don't seem to make a difference in occupancy. Also, why are these two highlighted genes of importance (pex7+, yhb1+), this is the first and last time they are mentioned?

    254-256: The statement that these TFs are indispensable may be too strong. Right before, the authors showed that most of these TFs don't change occupancy (and especially Fil1 and Reb1 do not show a correlation with up- and down-regulated genes, nor does Fil1 in FigS2B show a changed ChIP-seq signal).

    365: 'independently of the carbon source'. As far as we can see, all experiments were performed using glucose as the carbon source, so this statement seems too strong as there is no clear proof for this. This could be an easy extra experiment to perform these tests on media with other carbon sources than glucose?

    Fig1E: It is not clear if the experiment was performed with the Wild-Type or a deletion strain. In the case of the WT, colonies grew in the media not containing NSF but in Fig5E and Fig5I, the WT did grow in the media not containing NSF. It could be more relevant to plate out 1 colony like in the second screen. Thus, unless different strains were used for both experiments, the results seem inconsistent with each other, which is not mentioned in the manuscript.

    Fig1E, Fig5B, Fig5E, and Fig5I: For these experiments, different nitrogen concentrations were used depending on the media, but this has not been addressed/mentioned in the manuscript.

    Minor comments:

    53-57: I would like more elaboration on why CCR and NCR are important for virulence of human pathogens or relevant for industrial applications, and link back to this in the discussion. Otherwise, it is superfluous to include this in the introduction.

    108: Does having a h- library have any impact on the outcome compared to the original h+ library?

    170: Why would only one of the 117 NSF-linked genes change expression in the RNA-sequencing experiment? Any explanation as to why the expression remains unchanged for the 116 other NSF-linked genes?

    212: Please elaborate the discussion of these results. I understand the point that at low cell densities, the cells do not produce NSF as much, and thus adding NSF induces nrt1+. However, the added value of testing this in different media is unclear, especially when the results of strength of increase in nrt1+ show the opposite trend for the two different media between low and high nitrogen content.

    216: Why was the ADH1+ promoter chosen as a 'negative control'?

    284-288: Fig5E: To test whether AYR1 is indeed metabolizing NSF (and thus supporting this statement), an overexpression strain of AYR1 could be made to see if it grows on the EMM + NH4Cl without NSF added.

    385: 'NSF would not strictly revoke NCR only, but also CCR': the authors should try to provide experimental evidence, citation(s), or clearly state it to be a hypothesis. This comment links back to the major comment on line 365.

    405: typo: strains were validation, should be 'validated'

    644: typo: '+' sign not in superscript

    Fig1G: Could the differences in OCR be due to differences in growth rate or remaining glucose? It could for instance be that the culture in the control condition grew less fast, thus still having glucose and therefore still in fermentative metabolism. Showing or mentioning growth rates, nutrient concentrations could help to strengthen this finding.

    Fig2A: The top two 'most' upregulated genes (nrt1 and mei2) were taken along for additional experiments. However, one gene with a significant upregulation labeled in red on the left seemingly shows stronger induction than the second gene (mei2). Why was this gene not taken along?

    Fig2C: the x-axis label is not immediately clear to the reader.

    Fig4B: typo: 'non treatmentt'

    Significance

    This manuscript advances our understanding of nitrogen signaling pathways and nitrogen catabolite repression in the model organism S. pombe. Specifically, it shows how a nitrogen signaling factor functions to send signals and adapt the metabolism upon a change in nutrients and reveals that this nitrogen signaling factor triggers a change from fermentation to a respiratory metabolism. These findings are relevant for the broad fields of applied microbiology, signal transduction and metabolic regulation. Relevant literature is appropriately cited, although the links with Crabtree repression in S. cerevisiae are perhaps not fully supported.

    This manuscript was reviewed by experts with expertise in S. cerevisiae, Crabtree effect, respiration-fermentation balance, adaptation to changing environments.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    We have now reviewed the manuscript by the groups of Dr. Bühler and Dr Yashiroda entitled: "Nitrogen signaling factor triggers a respiration-like gene expression program". We have enjoyed the topic, the experiments and the science behind it all. The authors study here one part of the fission yeast 'quorum sensing'-like mechanism of counteracting NCR, mediated by the small molecule NSF: they identify pathways required to respond to NSF, and more specifically determine the mechanism by which NSF counteracts NCR: triggering respiration. This is a very interesting manuscript, with nicely executed experiments, and the topic is of great interest. Regarding the major comments, they are specific to the current data. The minor comments are questions raised in light of the present set of data, which should be appropriate for future research and future manuscripts.

    Major comments

    1. Consistency with the numbers of mutants/genes should be improved. Line 119: 203 genes, 206 in 4th datasheet of Table S1; line 139: 117 mutants but 119 analyzed for GO analysis (1st datasheet in Table S2); lines 179 and 181: where are these numbers (92 down and 156 up) coming from? (compare with 74 and 98 in line 167) (maybe they come from the merge of 2, 4 and 6 h, but it is not indicated).
    2. Lists of genes up- and down-regulated from the RNA seq data should be provided. GO terms are not useful. Add supplementary table, please.
    3. Comparing the transcriptomic response to that of Malecki et al 2020 in response to Antimycin A (EMBO Rep. 21:e50845) would be useful.
    4. The optical densities and whether NCR has been induced has to be clearly specified in each experiment. For instance, RNA seq data. Line 165: for the transcriptome experiment, NSF is added or not to low density cells (not indicated in results, figure legend nor materials and methods). Should addition of NSF to wild-type strain en MM trigger the same transcriptomic changes?
    5. Fig. 1G: Addition of NSF can enhance oxygen consumption at any cell density? And in prototrophs? And without NCR? Add in figure legend that this has been done at OD600 of 0.01.
    6. Fig. 1 E and F: why 14 d after growth there is not growth at 2% glucose in panel F, but it is 10 d after in panel E? What is EMM Biomedical?
    7. Fig. 2BC: Venn diagrams should be more useful to demonstrate overlap withn the Malecki data.
    8. Fig. 4A: not very useful
    9. Is Hsr1 required for some of the RNA seq changes upon NSF addition? Same with other TFs
    10. Line 287: '...it is tempting to speculate that Ayr1 dampens adaptive responses by metabolizing NSF'. Calculating MEC for NSF in delta ayr1 and in cells over-expressing Ayr1 would be required to confirm this speculation. According to Pombase, cells lacking Ayr1 have their respiratory functions compromised (no growth in galactose, glycerol...), why is so? The opposite should be expected, if NSF-mediated respiration is enhanced in this background.
    11. Regarding the two pull-down experiments, one to identify Ayr1 and the second Hmt2, why different negative controls are used? Is addition of NSF to WCE prior to pull-down also used in the second experiment (with delta ayr1 and AlkOle)?
    12. The data regarding Hmt2 is very interesting. As for delta ayr1, delta hmt2 cells cannot grow in glycerol nor galactose according to Pombase. Is the result shown in Fig. 5J (lack of NSF-dependent activation of nrt1 and mei2 in delta hmt2) a consequence of the absence of the NSF receptor, or is it due to the lack of respiration of this background? Is delta hmt2 really auxotrophic for Cys? Why? In this background, H2S should be enhanced, and Cys and Met biosynthesis improved. In fact, in one manuscript these cells grow fine in SG minimal media (Mol Microbiol 01 42:29), while another report indicates they are auxotrophic for Cys (Genes to Cells 2016, 21:530).
    13. M&M: regarding RNA isolation and sequencing: add info about OD of cultures, genotype (leu1-32?), growth media; also, number of replicates and filtering (fold-change used, Q value...)
    14. M&M, ChIP seq: same as above. Also, MACS2 can be used for the unbiased identification of bona fide TF targets, by using a quantification tool reporting percentage of occupancy upstream the TSS (callpeak function).

    Minor comments

    1. Who triggers NCR? Analysis of 137 genes in Figure 1b.
    2. Synthesis of NSF: how is it regulated, where does it come from?
    3. NCR impairs import of BCAA. How are the aa importers such as Cat1 or Agp3 eliminated from the plasma membrane? Transporter internalization, degradation, transcriptional repression... And how does NSF block the NCR regarding aa uptake? Or aa usage?
    4. How can enhanced respiration by NSF counteract all of the above? How can now leu1-32 cells grow?
    5. Addition of NSF to any cell type would do the same, enhance respiratory rates? With or without previous NCR? Should this signaling molecule also drive different respiratory rates in a cell density-independent manner regarding glucose catabolite repression?

    Significance

    Within this manuscript, the authors study a cell-to-cell communication process, by which nitrogen catabolite repression can be counteracted by a small molecule called NSF. Specifically, the authors demonstrate here that NSF up-regulates respiratory metabolism as a mechanism to overcome the repression of amino acid internalization, which was blocked by excess nitrogen. This is a wonderful manuscript, with splendid data, on a very interesting topic.

  5. Version published to 10.1101/2023.12.18.572203v1 on bioRxiv