Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries

ABSTRACT The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial workhorse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further toward industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidizing agent that binds free thiols and creates disulfide bonds between them, thereby causing a so-called “disulfide stress” in bacteria. The method shows promise to selectively identify phenotypes in B. subtilis with improved tolerance toward oxidative and disulfide-associated stress. Phenotypes initially identified by transposon mutagenesis were recreated through targeted gene deletions. Among the resulting deletion mutants, the largest difference in diamide tolerance compared to the parental strain was observed for pfkA and ribT deletion strains. A proteomics analysis showed that diamide tolerance can be achieved through different routes involving increased expression of stress management proteins and reduced availability or activity of the RNA degradosome. We conclude that our screening method allows the facile identification of Bacillus strains with improved oxidative stress tolerance phenotypes. IMPORTANCE During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

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Review Spectrum01608-23
This paper elucidate two aspect of interest in Bacillus subtilis biotechnology: the role and pleiotropic effects of PfkA and RibT on diamide induced stress, and theTn mutagenesis as a strategy to find new genes involved in disulfide stress, without previous knowledge of sequences or functions, and to plan strain improvements by seamless targeted mutagenesis.The role of these two functions in future could be confirmed by gain of function approaches.
The results suggest a role concerning of the NADPH pool and of the RNA degradosome in the Bacillus response to oxidative stress and open the possibility of strain improvement for industrial production.
The paper needs some revisions before publication, here listed:  [13][14] The sentence "The calculated ratios were binned, which is expected to result in a downward sloping curve.Indeed, such a curve was observed for the parental strain under control conditions (without diamide), where the largest group of proteins was oxidized…" is unclear; • Pg.14 "At 1 h after introduction of diamide, accumulation of proteins assigned to the following functional categories could be detected for all three strains (parental, ΔpfkA, ΔribT)..", but in Fig3 Supplementary materials also in the parental strain after 1 hr of diamide exposure the amount of proteins is significantly changed; • Pg. 16 Why Authors say that Tn mutagenesis was not part of the study.It was not the purpose of the described work, but was an essential part and a good methodological choice; • Pg.17In Discussion more comments are needed about the results observed concerning BrxB protein (see Pg.14).• Pgs.21, 22, 23 and others, each References citation need careful revisions from the editing point of view: see cit 15, names instead of family names for some Authors or for example cit 42 Saccharomyces that should be in capital.Everywhere use italics for the species names.

Minor revisions or editing problems:
• Pg.6 in Material and Methods the medium LB according to literature is Luria and Bertani Medium and not Lysogeny medium, see also your ref. 50; • Pg.6 and at pg.7, in Material and Methods, the paragraphs "An overnight culture in ….All plates were incubated at 37C" is repeated twice at pg. 6 under the title "Kinetic diamide growth assay" where it would be off topic and probably an unnecessary section; • Pg.7 in Materials and Methods, specify the experimental provenance of the "deep well plates stored at .."; • Pg. 8 in Materials and Methods, in differential cysteine labeling, the switch of labeling order of the third sample was done as a control?• Pg10 in Library generation and coverage, refer to Supplementary Table 2 not 1; 1 Manuscript No. Spectrum01608-23 'Diamide-based screening method for isola on of improved oxida ve stress tolerance phenotypes in Bacillus mutant libraries' (Walgraeve et al.)

Authors' response to the comments of the Reviewers
Please note that the comments by the two Reviewers were divided up and marked in black.Our responses are marked in blue.Please note also that all references to page and line numbers in our responses below relate to the 'Track Changes' version of our manuscript in which all revisions have been marked with the Track Changes tool of Word.

Author's response to the comments of Reviewer #1:
This paper elucidate two aspect of interest in Bacillus subtilis biotechnology: the role and pleiotropic effects of PfkA and RibT on diamide induced stress, and theTn mutagenesis as a strategy to find new genes involved in disulfide stress, without previous knowledge of sequences or functions, and to plan strain improvements by seamless targeted mutagenesis.The role of these two functions in future could be confirmed by gain of function approaches.The results suggest a role concerning of the NADPH pool and of the RNA degradosome in the Bacillus response to oxidative stress and open the possibility of strain improvement for industrial production.The paper needs some revisions before publication, here listed: Response: We thank the Reviewer for the construc ve comments and sugges ons, which have helped us to improve our manuscript.• Pg.10 Comment about the possibility that also a prolonged treatment with Diamide may cause "stress directed mutagenesis" and produce undesirable mutants.See Sung and Yasbin, JOURNAL OF BACTERIOLOGY, Oct. 2002, 184(20), 5641-5653; Response: We appreciate the comment of the Reviewer.Accordingly, we have addressed the need of avoiding the accumula on of undesired spontaneous muta ons that are not related to a transposon inser on on page 10 (lines 293-294).
• Pg.10 and Supplementary Table 2: the amount of viable screened CFU seems incoherent with the data in SupplementaryTable 2, possibly TMA1 and TMA4 viable amounts screened are +04?Response: In response to the comment of the Reviewer, we have rephrased this paragraph to avoid misunderstandings by the readers of our manuscript.It now reads as follows: "Mutant libraries were generated by four cycles of transposon mutagenesis, plated on LB agar containing 10 µg/mL kanamycin and 200 µM diamide, and incubated at 37 °C.Colonies were picked at 16 h, 24 h, and 40 h a er pla ng and transferred to fresh LB-kanamycin plates.Thus, a total of 312 candidate colonies of bacteria with poten ally increased diamide tolerance was selected.In parallel to the selec on of diamide tolerant transposon mutants, CFUs were counted on LB plates with kanamycin, but without diamide, in order to es mate the total number of bacteria with transposon muta ons (Supplementary table 2).These plates were incubated at 37 °C to avoid plasmid replica on.The results showed that an average viable count of 1.4 ± 0.7 x10 5 CFU/mL was obtained a er heat shock.With 100 µl aliquots plated on 97 plates, this implied that a total of about 1.4 ± 0.7 × 10 6 viable transposi on events were screened for diamide tolerance" (page 10, lines 302-311).
• Pg.10 5.85 x 105 is the number of estimated TA sites, Himar1 targets, in B. subtilis genome?If yes specify; Response: We originally es mated the theore cal number of TA sites available for transposon integra on by the length (4.2 Mb) and GC content (43.5%) of the B. sub lis genome, and by assuming a random distribu on of nucleo des.However, we have now determined that there are precisely 561325 TA sites in the B. sub lis 168 genome.This is now specified on page 10 (lines 312-314).
• Pg.13 and Supplementary Fig. 4: SigV regulon don't show, in presence of pfkA deletion, higher abundance proteins (red hexagon); moreover where are shown SigX and SigY regulons mentioned in Pg.13? Response: Indeed, we observed no induc on of SigV-regulated proteins in the p A dele on strain at 1 hour a er addi on of diamide.SigX-regulated proteins are shown on the le lower half of the Voronoi treemap.Indeed, there are no SigY-regulated proteins included as these proteins were not iden fied in our proteome analysis.We have corrected this in the manuscript (page 13, line 446).
• Pg. 13 "the percentage-wise rate of cysteine oxidation was determined for each protein.." or "for each protein sample"?Response: Indeed, the percentage-wise rate of cysteine oxida on was determined for each protein in each sample.We have now clarified this (page 13, lines 466-468).
• Pg. 13-14 The sentence "The calculated ratios were binned, which is expected to result in a downward sloping curve.Indeed, such a curve was observed for the parental strain under control conditions (without diamide), where the largest group of proteins was oxidized..." is unclear; Response: We thank the Reviewer for poin ng out that this sentence was unclear and have rephrased it as follows: "The calculated ra os were binned, which typically results in a distribu on where higher rates of thiol oxida on are increasingly less abundant.Such a distribu on was observed for the parental strain under control condi ons (without diamide), where the largest group of proteins was oxidized for less than 5% (Figure 8)" (pages 13-14, lines 468-478).
• Pg.14 "At 1 h after introduction of diamide, accumulation of proteins assigned to the following functional categories could be detected for all three strains (parental, ΔpfkA, ΔribT)..", but in Fig3 Supplementary materials also in the parental strain after 1 hr of diamide exposure the amount of proteins is significantly changed; Response: The Reviewer is right that also the proteome of the parental strain showed significant changes at 1 hour a er diamide addi on as shown in Supplementary figure 3.In fact, we had indicated this with the word "parental" in parenthesis.We have now made this more clear by specifying "parental strain" (page 14, line 494).Furthermore, we have made this more explicit by rephrasing lines 488-491 (page 14), which now read as follows: "This could be inferred from the experimental replicates.This has now been specified in the Methods sec on on page 9 (lines 269-270 and lines 272-274).
13. P.14-15 "Notably" all these statements are not supported by data so why include?Response: We appreciate this comment of the Reviewer, but actually all these data are presented in Supplementary table 3. To guide the reader to consult this table, we have now more regularly referred to it (see pages 14-15, lines 498, 505, 512, 524, 528, 533, 541 and 550).
14. P.16 7 lines up-what is the significance of the ribosomal protein changes?Is this reflected in rRNA as well?Response: The Reviewer raises an interes ng ques on, but in our present analysis we did not inves gate RNA levels.RNA analyses would certainly represent an interes ng follow-up of our present inves ga on.However, we believe that such analyses are beyond the scope of our present study, which was aimed at developing a diamide-based screening method for Bacillus strains with improved oxida ve stress tolerance (see page 5, lines 101-102).

P.19 line 4-what does "variable" mean and why?
Response: We agree with the Reviewer that the word "variable" was a bit vague in the present context and have rephrased the sentence.It now reads as follows: "The encountered phenotypes could be traced back to different genomic disrup ons and the mechanisms behind these phenotypes were also dis nct" (page 19, lines 672-674.16.Introduction-explain "more robust" and cite evidence. Response: What we meant to say with the words "more robust" is that our present screening method will enhance the development of industrial produc on strains with improved robustness under condi ons of oxida ve stress.Of note, we used the wording "more robust" only in the Importance paragraph of our manuscript.We have rephrased the respec ve sentence to clarify what we mean (page 3, lines 53-54).17.P.8 details in paragraphs 2&3 can be shortened.Response: In principle, we could shorten these paragraphs.However, we believe that it will be helpful for the readers of our manuscript to keep these paragraphs as they are in the present manuscript version.This will help the readers to appreciate at a glance what we have done exactly.We therefore prefer not to shorten the two paragraphs.In case the Editor prefers shortening, we shall of course be ready to do this.18. Do other oxidizing agents cause similar changes in designated genes or are they unique to diamide?Not clear what the value of resistant strains is and is there data for this?Would be valuable information to include.Response: The Reviewer raises a relevant point that we actually addressed in the Introduc on of our manuscript where we wrote the following: "Oxida ve stress can originate from a variety of sources and is caused by oxida ve damage of cellular components [10].An example of this is the oxida on of free thiol groups of cysteine residues in proteins [11].Molecules that can cause this type of stress are reac ve oxygen, nitrogen, chlorine, and electrophilic species.The oxida ve stress response generally refers to the adapta ons made by the cell to deal with the adverse effects caused by these oxidants

• Pg. 4
Add a review reference on examples of Tn mutagenesis to obtain different independent bacterial mutants; Response: As proposed by the Reviewer, we have included addi onal references on transposon mutagenesis in our revised manuscript (new Refs 18-20, page 4, line 81).