Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

  1. Stuart D. Woodcock
  2. Karl Syson
  3. Richard H. Little
  4. Danny Ward
  5. Despoina Sifouna
  6. James K. M. Brown
  7. Stephen Bornemann
  8. Jacob G. Malone

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Abstract

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

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  1. Version published to 10.1371/journal.pgen.1009524
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    Reply to the reviewers

    Response to reviewers: Woodcock et al. 2021

    Reviewer 1 (Evidence, reproducibility, and clarity):

    Summary* *The authors resolved the biosynthesis of trehalose and alpha-glucan in Pseudomonas aeruginosa and the role of these two compounds in osmotic and desiccation stress.

    We thank the reviewer for their positive review of our manuscript. Our responses to their specific queries are interspersed below.

    Major comments:

    • Are the key conclusions convincing? Yes
    • *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? * No
    • *Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. * Not necessary, comprehensive coverage of research topic.
      • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. * Not applicable
    • Are the data and the methods presented in such a way that they can be reproduced? Yes
      • Are the experiments adequately replicated and statistical analysis adequate? * Yes, everything is adequate but just one subtle concern: check the significance of the number of digits in the entries listed in Table S3. Revise Table S3.

    Table S3 has been revised as requested. The data in this table is now presented correct to one decimal place.

    Minor comments:

    • *Specific experimental issues that are easily addressable. * Not applicable (Table S3: see above)
      • Are prior studies referenced appropriately?* No. Refs. 18- 32: The subjects of 'trehalose' and 'osmotic stress' have already been addressed in the Pseudomonas field and should be referenced. The authors cite work carried out on trehalose and osmotic stress on phylogenetically distant microorganisms, but do not cite related work from the Pseudomonas field which I consider to be inappropriate. Similarly, trehalose biosynthesis in Pseudomonas has not only been covered by refs. 47 and 48.

    This is a fair comment. The focus of our introduction came from a desire to concentrate specifically on the metabolism and intracellular function of trehalose/α-glucan in Pseudomonas. In hindsight, we acknowledge that our introduction is a little too narrowly focussed. We have expanded the introduction and discussion sections to include additional discussion of trehalose in Pseudomonas and its regulation in the CF lung.

    • Are the text and figures clear and accurate? Extremely well written manuscript and prepared figures
      • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? *Revise the list of references and discuss more thoroughly your novel findings in the light of existing knowledge in the Pseudomonas field.

    Please see previous comment relating to the literature.

    Reviewer 1 (Significance):

    Significance

      • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.* Conceptual advance: The authors identified and characterized the enzymatic pathway of trehalose and alpha-glucan biosynthesis in Pseudomonas aeruginosa and its role to cope with osmotic and desiccation stress. The authors' conclusions do not correspond with recently published peers' work; hence they should discuss in more detail why they consider their data to be more accurate to discern the role of trehalose to contain desiccation and osmotic stress in P. aeruginosa.

    Please see previous comment relating to the literature. In general, the published work to date on trehalose in Pseudomonas spp. does not consider GlgE pathway-mediated link to α-glucan that we characterise in this paper. Our work demonstrates that synthesis and metabolism of the two molecules are implicitly linked in species where the GlgE pathway is present, and they cannot be considered in isolation. For this reason we are very confident that our study represents the most accurate model to date for trehalose and α-glucan metabolism and their associated phenotypes in P. aeruginosa. We have therefore emphasised that the role of trehalose in Pseudomonas spp. should be re-evaluated in light of our findings.

      • Place the work in the context of the existing literature (provide references, where appropriate).* Existing literature focusing on trehalose, osmotic stress, desiccation stress in the *Pseudomonas *field not cited by the authors:
    • These papers are of variable scientific quality, but the conceptual work by Hallsworth and the work by Behrens on the PA metabolome in CF lungs are worth discussing. All other work provides pieces of information on function and biosynthesis of trehalose up to now known by the Pseudomonas community. The authors resolved the function of the GlgA operon which will be definitely appreciated.

    We thank the reviewer for these helpful suggestions. We have reviewed these papers carefully and have incorporated several, including the papers from Hallsworth and Behrens into the revised manuscript.

    Strengths of the manuscript:

    • Meticulously planned and carefully executed experiments, not a single experimental flaw
    • Very high technical quality of experiments and primary data
    • Comprehensive coverage of the research topic
    • Excellent presentation in text and illustrations Only weakness:
    • Insufficient consideration of peers' published work on trehalose and its role in stress response in P. aeruginosa

    Please see previous comment relating to the literature.

      • State what audience might be interested in and influenced by the reported findings.* Scientists working in the fields of glycoconjugate and carbohydrate research, biochemists, microbiologists with interest in metabolic pathways, stress response and/or Pseudomonas. Reviewer #2 (Evidence, reproducibility, and clarity):

    It will be difficult for me to write a review of this paper and for the authors to make sense of my review because the manuscript's pages / lines are not numbered…

    We apologise to the reviewer for this oversight.

    __*Summary *__The authors carried out a comprehensive characterization of the metabolism of trehalose in Pseudomonas aeruginosa PA01, using techniques of biochemistry, reverse genetics, and bioinformatics. The main findings include that the disaccharide trehalose is synthesized in this organism from branched chain α-glucans and that the catabolism of trehalose proceeds via another disaccharide, maltose and is fed back into the synthesis of α-glucans. Trehalose and α-glucans have been implicated in conferring resistance to abiotic stresses in other organisms. The authors show that mutants that are blocked in the synthesis of trehalose are sensitive to high salinity but are normal with respect to their sensitivity to desiccation, whereas mutants impaired in the accumulation of α-glucans are sensitive to desiccation without being unduly sensitive to osmotic stress. These results indicate that trehalose and α-glucans have different roles in abiotic stress-tolerance.

    Major points

    This manuscript describes an impressive amount of careful work and presents new insights into the metabolism of trehalose, maltose, and α-glucans. However, the authors should address the following major comments before the paper is accepted.

    We thank the reviewer for their thorough and positive assessment of the manuscript. We address their specific points below.

    • Discussion: the authors state that "trehalose protects Pseudomonas ssp. against osmotic stress, most likely due to its role as a compatible solute." According to Table 2, P. aeruginosa grown in the medium of low osmolarity accumulated 0.13% trehalose per gram dry weight, i.e. ~4 μmol / g dry weight. Assuming that the dry weight / wet weight ratio of P. aeruginosa is the same as that of P. putida, which is ~1/3 (PMID: 6508285), the concentration of trehalose in the cells calculates to be ~2 mM. It is not plausible that trehalose could be significant as compatible solute at this low concentration.
    • One way out could be if the accumulation of this disaccharide were increased by osmotic stress. The authors should also measure the trehalose content of cells grown in medium containing 0.85 M NaCl. In case of positive results in this experiment, it would be interesting to determine the effects of osmotic stress on the levels of trehalose biosynthetic and catabolic enzymes, but this would not be necessary for the acceptance of the paper.

    This is a fair point. To address this, we measured the trehalose and maltose-1-phosphate levels for PA01 grown in the presence of 0.85 M NaCl. We saw a highly significant increase in the abundance of trehalose, compared to growth on standard M9 media. This strongly suggests that trehalose accumulates under conditions of osmotic stress as suggested by the reviewer. These new results have been added to the relevant sections of the manuscript (M&M, results, table 2 and discussion). The student (Danny Ward) who conducted these new experiments has been added to the author list.

    • However, there is also an extensive literature suggesting that trehalose has antioxidant functions e.g. PMID: 29241092 (the first paper that came up in Google search for "trehalose as antioxidant"). The authors should discuss this possible alternate role of trehalose.

    The reviewer is correct that trehalose has well-documented antioxidant functions in various species. We have modified the introduction to address this. To maintain the focus of our manuscript on bacteria we have used a different example to that suggested by the reviewer.

    • It is not described adequately in the Materials and Methods how the cellular contents of trehalose and maltose-1-phosphate (M1P) were determined.

    The Materials and Methods section has been revised to include more details of this method.

    • I found the growth curves in Figure 8, especially in panel B, to be uninterpretable. The authors should spread these data into more panels or use some other method to make them clearer.

    We have expanded the legend for Figure 8 to describe more fully what is going on in this figure. The results in Figure 8 are grouped according to the operons in which each set of genes is located. As such, the graphs contain unequal numbers of curves, with 8B containing the most and 8C only showing data for WT and ΔglgP.

    • The statement "The GlgA and GlgE proteins . . . enable two alternate mechanisms for linear α-glucan biosynthesis", which is echoed a number of times in the manuscript, seems to create the impression that there are two de novo pathways of synthesis of these polysaccharides. However, as shown in Figure 1, the GlgA pathway is the only route to the net synthesis of α-glucans, and GlgE is only part of a recycling pathway. Therefore, it cannot be true that "the vast majority of α-glucan accumulated by P. aeruginosa will be produced by GlgE".

    We have revised this section to further clarify what we mean when we state that the majority of α-glucan accumulated by P. aeruginosa will be produced by GlgE. Our data suggest that there is a big difference between the generation of α-glucan (conducted by both GlgA and GlgE) and its accumulation (flux through GlgA generated α-glucan is high, so only GlgE generated α-glucan can accumulate to generate large polymers).

    • The authors state that "MalQ disproportionates (sic) α-glucan with glucose to produce maltose." Figure 1 shows that GlgE uses an "acceptor", which I assume could be glucose. How is free glucose synthesized? Could cells grown on a non-carbohydrate as sole carbon source make free glucose?

    P. aeruginosa is able to carry out gluconeogenesis, so it can produce glucose from non-carbohydrate carbon sources if necessary.

    Our data show that GlgE acceptor preference gets lower as the acceptor molecule gets shorter. It is possible to detect GlgE activity without an acceptor. In this case we see a lag, implying M1P hydrolyses slowly at first and priming with glucose is also slow. Eventually however, the products get long enough for the reaction to take off. MalQ will work with DP2 or longer as the donor and DP1 or longer as the acceptor, moving one glucose unit at a time.

    • Pedantic point, but "disproportionation" means an oxidation-reduction reaction in which two identical molecules are used to produce two different molecules (https://en.wikipedia.org/wiki/Disproportionation). The reaction catalysed by MalQ does not involve electron transfer. Don't the authors mean that this enzyme is a glycosyl transferase?

    We have checked this, and our use of disproportionation in the manuscript is correct. The definition of disproportionation is any desymmetrizing reaction of the following type: 2 A → A' + A", and is not limited to redox reactions. MalQ carries out a reaction of this type when presented with a maltooligosaccharide.

    • The authors state that TreS had "a very high Km for trehalose (>100 mM)". In view of the low concentration of trehalose (Point 1, above), the physiological relevance of this suggested activity is questionable.

    See response to question 1 above. As trehalose levels are elevated under osmostress conditions this concern becomes less critical. It is of course true that conditions* in vitro* may not fully reflect cellular conditions and that this activity may be higher in vivo, but this is a general limitation of all protein biochemistry studies. The important point here is that trehalose synthase activity is detected for PA01 TreS.

    • Explain better what "predicted mean log10(CFU) means.

    The predicted mean refers to the value of log10(CFU) predicted by the statistical model we use. We have clarified this in the relevant sections of the manuscript.

    • Can the authors suggest how "α-glucan protects PA01 against desiccation"?

    Without further investigation we can only speculate as to how α-glucan confers desiccation tolerance in PA01. One possibility is that α-glucan functions as a hydrogel, like the exopolysaccharide alginate, trapping water molecules and slowing their evaporation. Alternatively, it may confer a structural role akin to that of trehalose, preventing the loss of cell integrity as water levels decrease. We now address these possibilities in the discussion.

    • Can P. aeruginosa metabolize exogenous trehalose or maltose? If the authors know either way, they should mention it. If they don't know, I am not suggesting that they should test this for this paper, but it would be interesting to know whether these compounds would induce the expression of the trehalose or maltose catabolic enzymes or repress the relevant biosynthetic enzymes. >P. aeruginosa is able to metabolise exogenous maltose and trehalose. While the experiments that the reviewer suggests are certainly interesting, in our view tre/glg gene regulation is beyond the scope of the current manuscript. This field is certainly worth investigating in the future, however.

    Minor points

    • First page under "Results": "phosphomutase" should be "phosphoglucomutase"?

    Changed as requested.

    • Discussion: insert "P. syringae" before "Pto".

    Changed as requested.

    • Materials and Methods: describe how ADP was quantified in the maltokinase assay.

    The materials and methods section has been updated as requested.

    Reviewer 2 (Significance):

    Significance

    Until this work, the biosynthesis of trehalose has been most extensively characterized in Escherichia coli, in which it has been shown that this disaccharide is made by the reaction of glucose-6-phosphate and UDP-glucose to give trehalose-6-phosphate and dephosphorylation to trehalose, catalysed by OtsA and OtsB. The authors discovered a very different pathway in P. aeruginosa in which the synthesis of trehalose goes through α-glucans as intermediates.

    Because trehalose and α-glucans are needed for osmotic stress- and desiccation-tolerance, respectively, this work is of significance to researchers studying abiotic stress resistance.

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    Referee #2

    Evidence, reproducibility and clarity

    Review of manuscript "Trehalose and α-glucan mediate distinct abiotic responses in Pseudomonas aeruginosa" by S. D. Woodcock et al.

    It will be difficult for me to write a review of this paper and for the authors to make sense of my review because the manuscript's pages / lines are not numbered. I will do my best write a review, but for the future, I urge this Journal to print the text on pages in which the lines are numbered or require this of the authors.

    Summary.

    The authors carried out a comprehensive characterization of the metabolism of trehalose in Pseudomonas aeruginosa PA01, using techniques of biochemistry, reverse genetics, and bioinformatics. The main findings include that the disaccharide trehalose is synthesized in this organism from branched chain α-glucans and that the catabolism of trehalose proceeds via another disaccharide, maltose and is fed back into the synthesis of α-glucans. Trehalose and α-glucans have been implicated in conferring resistance to abiotic stresses in other organisms. The authors show that mutants that are blocked in the synthesis of trehalose are sensitive to high salinity but are normal with respect to their sensitivity to desiccation, whereas mutants impaired in the accumulation of α-glucans are sensitive to desiccation without being unduly sensitive to osmotic stress. These results indicate that trehalose and α-glucans have different roles in abiotic stress-tolerance.

    Major points.

    This manuscript describes an impressive amount of careful work and presents new insights into the metabolism of trehalose, maltose, and α-glucans. However, the authors should address the following major comments before the paper is accepted.

    1. Discussion: the authors state that "trehalose protects Pseudomonas ssp. against osmotic stress, most likely due to its role as a compatible solute." According to Table 2, P. aeruginosa grown in the medium of low osmolarity accumulated 0.13% trehalose per gram dry weight, i.e. ~4 μmol / g dry weight. Assuming that the dry weight / wet weight ratio of P. aeruginosa is the same as that of P. putida, which is ~1/3 (PMID: 6508285), the concentration of trehalose in the cells calculates to be ~2 mM. It is not plausible that trehalose could be significant as compatible solute at this low concentration.
      One way out could be if the accumulation of this disaccharide were increased by osmotic stress. The authors should also measure the trehalose content of cells grown in medium containing 0.85 M NaCl. In case of positive results in this experiment, it would be interesting to determine the effects of osmotic stress on the levels of trehalose biosynthetic and catabolic enzymes, but this would not be necessary for the acceptance of the paper.
      However, there is also an extensive literature suggesting that trehalose has antioxidant functions e.g. PMID: 29241092 (the first paper that came up in Google search for "trehalose as antioxidant"). The authors should discuss this possible alternate role of trehalose.
      It is not described adequately in the Materials and Methods how the cellular contents of trehalose and maltose-1-phosphate (M1P) were determined.
    2. I found the growth curves in Figure 8, especially in panel B, to be uniterpretable. The authors should spread these data into more panels or use some other method to make them clearer.
    3. The statement "The GlgA and GlgE proteins . . . enable two alternate mechanisms for linear α-glucan biosynthesis", which is echoed a number of times in the manuscript, seems to create the impression that there are two de novo pathways of synthesis of these polysaccharides. However, as shown in Figure 1, the GlgA pathway is the only route to the net synthesis of α-glucans, and GlgE is only part of a recycling pathway. Therefore, it cannot be true that "the vast majority of α-glucan accumulated by P. aeruginosa will be produced by GlgE".
    4. The authors state that "MalQ disproportionates (sic) α-glucan with glucose to produce maltose." Figure 1 shows that GlgE uses an "acceptor", which I assume could be glucose.
      How is free glucose synthesized? Could cells grown on a non-carbohydrate as sole carbon source make free glucose? Pedantic point, but "disproportionation" means an oxidation-reduction reaction in which two identical molecules are used to produce two different molecules (https://en.wikipedia.org/wiki/Disproportionation). The reaction catalyzed by MalQ does not involve electron transfer. Don't the authors mean that this enzyme is a glycosyl transferase?
    5. The authors state that TreS had "a very high Km for trehalose (>100 mM)". In view of the low concentration of trehalose (Point 1, above), the physiological relevance of this suggested activity is questionable.
    6. Explain better what "predicted mean log10(CFU) means.
    7. Can the authors suggest how "α-glucan protects PA01 against desiccation"?
    8. Can P. aeruginosa metabolize exogenous trehalose or maltose? If the authors know either way, they should mention it. If they don't know, I am not suggesting that they should test this for this paper, but it would be interesting to know whether these compounds would induce the expression of the trehalose or maltose catabolic enzymes or repress the relevant biosynthetic enzymes.

    Minor points.

    1. First page under "Results": "phosphomutase" should be "phosphoglucomutase"?
    2. Discussion: insert "P. syringae" before "Pto".
    3. Materials and Methods: describe how ADP was quantified in the maltokinase assay.

    Significance

    Significance.

    Until this work, the biosynthesis of trehalose has been most extensively characterized in Escherichia coli, in which it has been shown that this disaccharide is made by the reaction of glucose-6-phosphate and UDP-glucose to give trehalose-6-phosphate and dephosphorylation to trehalose, catalyzed by OtsA and OtsB. The authors discovered a very different pathway in P. aeruginosa in which the synthesis of trehalose goes through α-glucans as intermediates.
    Because trehalose and α-glucans are needed for osmotic stress- and desiccation-tolerance, respectively, this work is of significance to researchers studying abiotic stress resistance.

    The Reviewers' guidelines stipulate that Reviewers should define their fields of expertise.

    My credentials are: a) I have been solicited to review this paper, and b) I have publications in osmotic stress adaptation and trehalose biosynthesis in Enterobacteriaceae.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

    The authors resolved the biosynthesis of trehalose and alpha-glucan in Pseudomonas aeruginosa and the role of these two compounds in osmotic and desiccation stress.

    Major comments:

    • Are the key conclusions convincing?

    yes

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    no

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Not necessary, comprehensive coverage of research Topic

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Not applicable

    • Are the data and the methods presented in such a way that they can be reproduced?

    yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes, everything is adequate but just one subtle concern: check the significance of the number of digits in the entries listed in Table S3. Revise Table S3.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    not applicable (Table S3: see above)

    • Are prior studies referenced appropriately?

    No. Refs. 18- 32: The subjects of 'trehalose' and 'osmotic stress' have already been addressed in the Pseudomonas field and should be referenced. The authors cite work carried out on trehalose and osmotic stress on phylogenetically distant microorganisms, but do not cite related work from the Pseudomonas field which I consider to be inappropriate. Similarly, trehalose biosynthesis in Pseudomonas has not only been covered by refs. 47 and 48.

    • Are the text and figures clear and accurate?

    Extremely well written manuscript and prepared figures

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Revise the list of references and discuss more thoroughly your novel findings in the light of existing knowledge in the Pseudomonas field

    Significance

    2. Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Conceptual advance: The authors identified and characterized the enzymatic pathway of trehalose and alpha-glucan biosynthesis in Pseudomonas aeruginosa and its role to cope with osmotic and desiccation stress. The authors' conclusions do not correspond with recently published peers' work, hence they should discuss in more detail why they consider their data to be more accurate to discern the role of trehalose to contain desiccation and osmotic strass in P. aeruginosa.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    Existing literature focusing on trehalose, osmotic stress, desiccation stress in the Pseudomonas field not cited by the authors

    Pazos-Rojas LA, Muñoz-Arenas LC, Rodríguez-Andrade O, López-Cruz LE, López- Ortega O, Lopes-Olivares F, Luna-Suarez S, Baez A, Morales-García YE, Quintero- Hernández V, Villalobos-López MA, De la Torre J, Muñoz-Rojas J. Desiccation- induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy. PLoS One. 2019 Jul 19;14(7):e0219554. doi:10.1371/journal.pone.0219554.

    Wang T, Jia S, Dai K, Liu H, Wang R. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose. Can J Microbiol. 2014 Sep;60(9):599-604. doi: 10.1139/cjm-2014-0330.

    Harty CE, Martins D, Doing G, Mould DL, Clay ME, Occhipinti P, Nguyen D, Hogan DA. Ethanol Stimulates Trehalose Production through a SpoT-DksA-AlgU-Dependent Pathway in Pseudomonas aeruginosa. J Bacteriol. 2019 May 22;201(12):e00794-18. doi: 10.1128/JB.00794-18.

    Cross M, Biberacher S, Park SY, Rajan S, Korhonen P, Gasser RB, Kim JS, Coster MJ, Hofmann A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa. FASEB J. 2018 Oct;32(10):5470-5482. doi: 10.1096/fj.201800500R.

    Wang T, Jia S, Dai K, Liu H, Wang R. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose. Can J Microbiol. 2014 Sep;60(9):599-604. doi: 10.1139/cjm-2014-0330.

    Behrends V, Ryall B, Zlosnik JE, Speert DP, Bundy JG, Williams HD. Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections. Environ Microbiol. 2013 Feb;15(2):398-408. doi: 10.1111/j.1462-2920.2012.02840.x

    Behrends V, Ryall B, Wang X, Bundy JG, Williams HD. Metabolic profiling of Pseudomonas aeruginosa demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance. Mol Biosyst. 2010 Mar;6(3):562-9. doi: 10.1039/b918710c.

    Matthijs S, Koedam N, Cornelis P, De Greve H. The trehalose operon of Pseudomonas fluorescens ATCC 17400. Res Microbiol. 2000 Dec;151(10):845-51. doi: 10.1016/s0923-2508(00)01151-7.

    van der Werf MJ, Overkamp KM, Muilwijk B, Koek MM, van der Werff-van der Vat BJ, Jellema RH, Coulier L, Hankemeier T. Comprehensive analysis of the metabolome of Pseudomonas putida S12 grown on different carbon sources. Mol Biosyst. 2008 Apr;4(4):315-27. doi: 10.1039/b717340g.

    Hallsworth JE, Heim S, Timmis KN. Chaotropic solutes cause water stress in Pseudomonas putida. Environ Microbiol. 2003 Dec;5(12):1270-80. doi: 10.1111/j.1462-2920.2003.00478.x.

    Ball P, Hallsworth JE. Water structure and chaotropicity: their uses, abuses and biological implications. Phys Chem Chem Phys. 2015 Apr 7;17(13):8297-305. doi: 10.1039/c4cp04564e

    Cray JA, Russell JT, Timson DJ, Singhal RS, Hallsworth JE. A universal measure of chaotropicity and kosmotropicity. Environ Microbiol. 2013 Jan;15(1):287-96. doi: 10.1111/1462-2920.12018.

    Chin JP, Megaw J, Magill CL, Nowotarski K, Williams JP, Bhaganna P, Linton M, Patterson MF, Underwood GJ, Mswaka AY, Hallsworth JE. Solutes determine the temperature windows for microbial survival and growth. Proc Natl Acad Sci U S A. 2010 Apr 27;107(17):7835-40. doi: 10.1073/pnas.1000557107.

    These papers are of variable scientific quality, but the conceptual work by Hallsworth and the work by Behrens on the PA metabolome in CF lungs are worth discussing. All other work provides pieces of information on function and biosynthesis of trehalose up to now known by the Pseudomonas community. The authors resolved the function of the GlgA operon which will be definitely appreciated.

    Strengths of the manuscript:

    • Meticulously planned and carefully executed experiments, not a single experimental flaw • very high technical quality of experiments and primary data • comprehensive coverage of the research topic • excellent presentation in text and illustrations

    only weakness: • insufficient consideration of peers' published work on trehalose and its role in stress response in P. aeruginosa

    • State what audience might be interested in and influenced by the reported findings.

    Scientists working in the fields of glycoconjugate and carbohydrate research, biochemists, microbiologists with interest in metabolic pathways, stress response and/or Pseudomonas

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Reviewer's expertise: Pseudomonas genomics and physiology, respiratory tract infections, solid background in biochemistry and molecular biology

  8. Version published to 10.1101/2020.10.23.351924v1 on bioRxiv