PREreview of The broad-spectrum RumC1 bacteriocin targets a transient peptidoglycan intermediate of the nascent cell wall

The manuscript by Boyeldieu et al. explores the cell-wall poisoning mechanism of the RumC1 bacteriocin, produced by Ruminococcus gnavus E1. The authors developed an ordered genome-wide mutagenesis strategy in Streptococcus pneumoniae to identify RumC1-resistant mutants. These studies helped confirm RumC1's inhibition of transpeptidase activity, thereby blocking peptidoglycan (PG) synthesis and cell growth. The selective binding of RumC1 to the nascent PG had not been reported before. They also identified the immunity protein RumIc1 from the RumC1 biosynthetic gene cluster as sufficient to counteract RumC1 toxicity in S. pneumoniae. The findings reported in this study have exciting implications for antimicrobial resistance, potentially shifting the paradigm for treating infections caused by multidrug-resistant pathogens. Overall, the data in this manuscript is well presented and supports the authors' claims. We enjoyed reading this manuscript and outline major and minor adjustments to improve clarity in reporting and presentation, as well as providing additional context for a broader audience.

Major comments

- Figure 1D shows the morphology and HADA incorporation in WT and Rum1C-resistant mutants. In lines 206-208, you mention that Rum1C-resistant mutants show reduced HADA labeling; it would be helpful to include fluorescence quantification, as represented in Figure 6. Similarly, for Figures 2C and D, consider incorporating the quantification for cell width. It will also help support the results mentioned in lines 251-258. For instance, quantification of cellular morphology would provide clear evidence of “effects of RumC1 on cell morphology and PG synthesis differ from those caused by vancomycin”.

- To strengthen the conclusion that variants of RumIc1 do not confer immunity, consider including evidence that the protein variants are stable by showing an immunoblot. Along this line, please provide immunoblots for the strains used in Figure 4 and onwards, in which genes are expressed from an ectopic promoter site.

Minor comments

-All figures: Consider color coding the name of the strain and applying this color-coding across figures to improve reader comprehension.

-Ensure all supplementary figures are referred to in the main text.

-Statistic data representation: Consider changing “,” to “.” for the p values.

-The introduction is well written. A description of WalR and WalK and how they play a role in PG homeostasis would benefit non-initiated readers.

-RumC1 exhibits antibacterial efficacy against monoderm and LPS-poor strains, and an unencapsulated strain of Spn was used for this study. We understand how difficult it would have been to use an encapsulated strain for this work as they are more genetically recalcitrant, but clinically all pathogenic strains have a capsule. Please consider including discussion on the potential impact of RumC1 on an encapsulated strain.

-Figure 1F: The controls were appropriate and nicely contextualizes RumC1 stress induction; Bacitracin targets undecaprenyl pyrophosphate (membrane carrier for cell wall precursors) which serves as a positive control and kanamycin, which targets protein synthesis, serves as a negative control. Please consider including this information in the legend.

-Line 272: A little more background is needed about peptidoglycan (PG) synthesis, especially before mentioning the nascent PG.

-Figure 4: At 0.4 µM RumC1, explain why rumIc2 is having a lag? Why are MIC and growth curves not corresponding?

-Figure S9: How was the highest immunity achieved when all the immunity proteins were co-expressed? How did RumI_c2 become neutral?

-Figure 5C: Comment on the trend of growth of the strains at 0.4 µM RumC1 after 16 h, it appears that strains have started to grow again, likely because of suppressors. Consider including the growth curve for 24 h for clarity.

-Figure 6F: Please explain how the cleavage sites from MD simulation were predicted.

-Adding a few sentences in the discussion of how much RumC1 may be produced in the gut would be of interest, as well as speculation to what its effect on the gut microbiome may be.

- Figure 1C: Consider modifying the color of the control (WT) so it’s clearly apparent that 0.6 µM RumC1 results in no growth.

-Figure 1D: WT has debris; consider picking another field or enhancing the brightness.

-Figure 1E: spr1875 appears to be a little stretched in the X axis.

-Figure S3D: Please consider using in-text citations of Figure S3C and Figure S3D. Moreover, please provide an explanation of Figure S3D, in which the WT strain, when incubated with RumC1, shows lower expression of pcsB and spr1875 or is it a mislabeling?

-Figure 6A: Consider color coding the last 2 D-Alanine in the model where the cleavage is happening.

-Line 290: Consider defining sacculus and protoplast.

-Lines 121, 141, 252: Missing references.

-Line 200: Fluorescent microscopy should be changed to fluorescence microscopy.

-Line 410: Points mutants should be changed to point mutants.

-Figure S4: Please consider including the time of treatment on the graph for clarity.

-Line 231: Refer to Figure S5C to improve clarity.

-Figure 6C: This panel is not mentioned in the main text.

Josy Joseph and Tahreem Zaheer (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Carter Collins, Camy Guenther and Lily Pumphrey. We are part of the Dept. of Biology where Malcolm E. Winkler’s group is located. Malcolm is a co-author on a publication in which one of the authors (Cécile Morlot) provided antibodies; this prior interaction did not influence the choice of this preprint for our class.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.