Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant

ABSTRACT The conserved protein WhiA is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motif and is a transcription regulator of the key cell division gene ftsZ in actinomycetes. In Bacillus subtilis, the absence of WhiA influences both cell division and chromosome segregation; however, the protein does not regulate any gene involved in these processes. In this study, we addressed three alternative mechanisms by which WhiA might exert its activity in B. subtilis and examined whether WhiA influences either (i) central carbon metabolism, (ii) fatty acid composition of the cell membrane, or (iii) chromosome organization. Mutations in glycolytic enzymes have been shown to influence both cell division and DNA replication. To measure the effect of WhiA on carbon metabolism, we tested different carbon sources and measured exometabolome fluxes. This revealed that the absence of WhiA does not affect glycolysis but does influence the pool of branched-chain fatty acid precursors. Due to the effect of WhiA on chromosome segregation, we examine chromosome organization in a ∆whiA mutant using chromosome conformation capture (Hi-C) analysis. This revealed a local reduction in short-range chromosome interactions. Together, these findings provide new avenues for future research into how this protein works in the non-actinomycete firmicutes. IMPORTANCE WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

In general the experiments appear to have been carefully conducted and the text is mostly comprehensibly written, but needs further editing in language and style.My main concern however, is the fact that the experimental data is very rich, but there are few positive results.The results showed that WhiA neither functions by regulating central carbon metabolism nor affects fatty acid composition.Only chromosome conformation capture (Hi-C) analysis indicated short range interactions are reduced in the absence of WhiA.In order to reveal the mechanism of WhiA in B. subtilis, it is suggested that the authors should conduct indepth research from the perspective of compaction of the chromosome.
Reviewer #2 (Comments for the Author): The study by Bohorquez et al uses whole cell investigative experiments to understand the function of whiA in Bacillus subtilis.They identify a cell division and DNA segregation defect in zapA/whiA mutant in minimal media, changes in BCFA precursor levels using metabolomics, changes in transcriptomics that did not correlate with metabolome data, changes in fatty acid composition which does not translate to changes in membrane fluidity, and a reduction in short range chromosome interactions.This study highlights the complexity of whole cell studies to understand the function of single gene, as well as the interconnectedness of metabolism, DNA replication and segregation, and cell division.The study is well presented and logically explained, but requires a few modifications.Feedback: Figure 2: colorised microscopy images do not show fluorescent membrane labelling, they show phase contrast image pseudocoloured red, with DAPI in cyan.Images need to be corrected, or description updated.Knockdown of whiA in zapA deletion strain -did you confirm changes in expression?(Western blot or qPCR) Similarly, was there any polar effects on glmR and crh when whiA was knocked down?In the zapA mutant, chromosomal defects and minicells observed when whiA depleted -does FtsZ localise over chromosomes in this double mutant?Are Z rings misplaced?The membrane fluidity assay described is in methods and discussed in results, but no data is included.This could be included in Figure 7.The data for the growth rescue of whiA mutant when BCFA are added is also not shown -this could be a supplementary figure, or stated that data is not included.Was this growth rescue performed in LB? Methods -details lacking for microscopy section that needs to be expanded.There is no reference for concentrations and incubation times for fluorescent probe labelling.Do you mean FM-595 was used to label membranes?Information about transmitted light method is missing -presumably phase contrast was used?Information about objective magnification and numerical aperture is missing.No details on image analysis are included -how were cell lengths and internucleoid distances measured?Manually?What parameters were used?Minor comments: Line 25 -spelling: motif Figure 1 -graphs show both OD600 and OD500 -is this correct?Lines 110, 115 and 116 reference Figure 1A but no alphabetical labelling is included in Figure 1 Lines 308 and 309 -Do you mean µM for FA concentrations?

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Point-by-point response to reviewers comments
We would like to begin by thanking the reviewers for their efforts and useful comments.For clarity, our replies are presented in blue.
Reviewer #1 (Comments for the Author): In general the experiments appear to have been carefully conducted and the text is mostly comprehensibly written, but needs further editing in language and style.My main concern however, is the fact that the experimental data is very rich, but there are few positive results.The results showed that WhiA neither functions by regulating central carbon metabolism nor affects fatty acid composition.Only chromosome conformation capture (Hi-C) analysis indicated short range interactions are reduced in the absence of WhiA.In order to reveal the mechanism of WhiA in B. subtilis, it is suggested that the authors should conduct in-depth research from the perspective of compaction of the chromosome.
We agree with the reviewer that part of our results describe the disproval of two working models for the conserved regulator WhiA (a role in central carbon metabolism and regulation of fatty acid composition).However, this information is important since these were plausible working models, and therefore helps to focus the effort to elucidate the working mechanism of WhiA.In that sense, the disproval of these two working models can be considered positive results.In addition, we do provide a lead for further research and that is a role of WhiA in the organization of the chromosome.Of course, this now needs to be further investigated in detail.Unfortunately, the tools to study chromosome organization at the next level are not trivial, and will require a whole new PhD/postdoc project, that goes beyond the current project.We hope we can perform such in-depth study in the future.
Reviewer #2 (Comments for the Author): The study by Bohorquez et al uses whole cell investigative experiments to understand the function of whiA in Bacillus subtilis.They identify a cell division and DNA segregation defect in zapA/whiA mutant in minimal media, changes in BCFA precursor levels using metabolomics, changes in transcriptomics that did not correlate with metabolome data, changes in fatty acid composition which does not translate to changes in membrane fluidity, and a reduction in short range chromosome interactions.This study highlights the complexity of whole cell studies to understand the function of single gene, as well as the interconnectedness of metabolism, DNA replication and segregation, and cell division.The study is well presented and logically explained, but requires a few modifications.

Feedback:
Figure 2: colorised microscopy images do not show fluorescent membrane labelling, they show phase contrast image pseudo-coloured red, with DAPI in cyan.Images need to be corrected, or description updated.
We would like to thank the reviewer for pointing out this mistake.Indeed the red background in the images is not membrane stain but pseudocolored phase contrast images to increase the contrast with the DAPI stained nucleoids.We have now indicated this clearly in the legend of Fig. 2 (lines 618-625 in the revision).
Knockdown of whiA in zapA deletion strain -did you confirm changes in expression?(Western blot or qPCR) Similarly, was there any polar effects on glmR and crh when whiA was knocked down?
The WhiA depletion strain has been extensively documented in previous publications (Surdova et al. 2013PMID: 24097947, Bohorquez et al. 2028 PMID: 29378890), and we have not checked again this depletion.From the previous work we know that it takes some time before morphological changes, such as cell elongation, become visible, as we also have observed in this study and show in Fig. 2A and 2B, and have mentioned in the text.In addition, in the Surdova et al. 2013 PMID: 24097947 paper we have extensively checked that the cell division phenotype was not related to any polar effect on crh.We have added this information now to the main text (line 128 in the revision).With respect to glmR, this gene is located upstream of whiA and therefore not affected by the Pspac insertion used in the WhiA depletion strain (Surdova et al. 2013 PMID: 24097947).This misunderstanding is possibly a consequence of the fact that we have not clearly explained the organization of the operon.We do this now in line 101 of the revision.
In the zapA mutant, chromosomal defects and minicells observed when whiA depleted -does FtsZ localise over chromosomes in this double mutant?Are Z rings misplaced?Upon depletion of WhiA in a zapA mutant cells become elongated and Z rings disappear.We have shown this in Surdova et al. 2013 PMID: 24097947.But, we agree that it would be good to mention this clearly in the text and we have added this information in line 120 of the revision.
The membrane fluidity assay described is in methods and discussed in results, but no data is included.This could be included in Figure 7.
We present the Laurdan generalized polarization results, used to measure membrane fluidity, now in the new Table S4 (referred to in line 257 of the revision).Since the Laurdan GP values did not differ between wild type cells and the whiA mutant, we feel this data is better placed in the SI.
The data for the growth rescue of whiA mutant when BCFA are added is also not shown -this could be a supplementary figure, or stated that data is not included.Was this growth rescue performed in LB?
We mention now in the main text "data no included" (line 259 in the revision), and indicate that the BCFA precursor were added to the LB medium (line 258 in the revision).
Methods -details lacking for microscopy section that needs to be expanded.There is no reference for concentrations and incubation times for fluorescent probe labelling.Do you mean FM-595 was used to label membranes?Information about transmitted light method is missing -presumably phase contrast was used?Information about objective magnification and numerical aperture is missing.No details on image analysis are included -how were cell lengths and internucleoid distances measured?Manually?What parameters were used?
We have now provided more details on the microscopy techniques used, including, the concentration of the fluorescent membrane and DNA dyes, incubation time and details on the microscopic setup (lines 320-327 of the revised manuscript).
Line 25 -spelling: motif Thank you for pointing this out.The spelling mistake has been corrected.
Figure 1 -graphs show both OD600 and OD500 -is this correct?Thank you for pointing this out.It should have been OD 500 for both graphs.We have now corrected this, and also explained in the Material and Methods when we use OD500 and OD600 (line 308 in the revision).
Lines 110, 115 and 116 reference Figure 1A but no alphabetical labelling is included in Figure 1 Thank you for pointing out this typo.No alphabetical labelling is necessary in Fig. 1.We have now removed this from the text (lines 110, 115 and 116 in the revision).
Lines 308 and 309 -Do you mean µM for FA concentrations?Thank you for pointing out this typo.Yes indeed it should have been µM.We have now corrected this (lines 310 and 312 in the revision).Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred -23R1 (Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant) Dear Prof. Leendert W. Hamoen: