Peer review of: Prominent members of the human gut microbiota express endo-acting O-glycanases to (2020)




REVIEWERS' COMMENTS: 

Reviewer #2 (Remarks to the Author): 

This revised manuscript by Crouch et al. is significantly better than the original submission. The 
significance and discussion is now much better conveyed, and the conclusions are appropriate for 
the study. The authors have also sufficiently addressed the reviewers' comments and suggestions. 

Reviewer #3 (Remarks to the Author): 

In this revision, the authors have effectively addressed nearly all of my previous comments, 
resulting in a much improved manuscript. In particular, the logical flow of the text is significantly 
better and easier to follow, and the correction of numerous errors and omissions in the data 
presented greatly improves confidence in the analysis. I only have a few minor comments on this 
revision: 

1. Regarding my original comment on the multiple sequence analysis and phylogeny, "Please 
provide the input multiple sequence alignment files in FASTA format and trees in a suitable format 
(e.g. Newick, PhyloXML) as supplemental information files to allow reader direct access to the 
underlying data." 

- I had requested that the authors provide their *alignment* files used as input for the 
phylogenetic analysis (i.e. the CLUSTAL output, which should contain alignment gaps), but it 
seems that the two SI files provided with this revision are the input files for the CLUSTAL 
alignment. The point here was to allow reader to examine the alignment quality, on which the 
phylogeny is dependent. Please provide the CLUSTAL output files in FASTA format (as a simple 
text file, if possible, not a Word file). 

- I do not see tree files included among the SI files in the revision. Please provide them. 

- Lastly, bootstrap values should be indicated on the published trees, at least above a certain 
statistical value, e.g. 50%. I appreciate that the authors were unable to run the usual 100 ML 
bootstraps due to computing limitations, but some indication of branch robustness should be 
provided in the figures. 

2. lines 111-114: Given that a number of GH16 members specifically from Bacteroides species 
have been characterized with porphyranase, agarase, beta1,3 glucanase and mixed-linkage 
glucanase activities, a statement to this effect and citation of the primary literature would be 
helpful for context (PMIDs 23150581, 29535379, 32265336, 29020628). 

3. lines 332-334 (also to provide context for line 444): Please provide supporting primary 
references here, especially to functionally and structural characterized GH16 subfamily 3 enzymes 
(http://www.cazy.org/GH16_characterized.html). Also, I believe line 334 should say "mixed 
linkage glucan". 

4. line 350: Do the authors wish to refer to the +1 subsite, specifically? 

5. line 356: "its" (not "it's" = it is) 

6. line 390: "was" 

6. lines 390-393: While I appreciate that the authors are being justifiably cautious here in avoiding 
prescribing specific biomarkers, it would seem to be valuable to future researchers to quantify the 
available HPLC data in a table. The chromatograms in Fig. 7 show some very obvious differences 

between samples, e.g. the predominance of peaks from (2) in panel a, varying amounts of (13) 
across all samples, etc. Using the existing HPLC data, please provide an SI table of peak areas for 
all glycans identified. Ditto for the data in SI figure 21. 

7. Figures 1 and 3: Please include the linkage key here also. 

8. SI figures 17 and 18. The last sentence of the corresponding legends appears to be incomplete 
or grammatically incorrect. 

End of comments.