Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This important review article provides a critical assessment of recent advances in the decoding of bacterial N-terminal proteoforms, which embodies a complexity that challenges established gene annotation. The vast number and types of N-terminal proteoforms provide a level of heterogeneity which impacts phenomena including  protein localization and stability, molecular interactions  and multiprotein complex assembly. The developments presented here include advances that continue to drive N-terminal proteoform discovery as well as their functional characterization, while highlighting approaches to their formation that enable selective manipulation of alternative translation initiation events.

While specific modifications of protein N-termini addressed in this review include acetylation, phosphorylation, glycosylation, addition of ubiquitin-like proteins and lipidation, no mention is made of the well-described process in which lipid membrane anchors are affixed to the N-termini of tetraheme cytochromes c that are associated with bacterial photochemical reaction centers. These lipid anchors consist of a diacylglycerol [https://doi.org/10.1021/bi00384a036, https://www.nature.com/articles/s41586-018-0014-5] in thioester linkage with a newly-exposed N-terminal Cys residues arising from signal peptide removal, as well as a triacyl structure [doi: 10.1038/s41586-018-0002-9] made up of an N-acyl chain and an S-diacylglycerol also bonded to a newly-exposed N-terminal Cys. This is reminiscent of the triacylation observed for the E. coli lipoprotein CusC (https://doi.org/10.1371/journal.pone.0015610), which provides an accessory linkage for this outer membrane localized heavy metal efflux pump to the cytoplasmic membrane. Moreover, regulatory components for this system have been described (https://doi.org/10.1016/j.isci.2025.113715). These various N-terminal lipidation phenomena deserve mention in this otherwise very comprehensive review.

Typos and other text errors detected:

L. 11: rather than simply a static….

L. 12: research has revealed…..

L. 119: Sentence represents a direct repeat of previous sentence.

L. 146, here and elsewhere: typhimurium

L. 223: Change order of sentence to coincide with A, B and C designations of Fig. 3.

Fig. 3A: rpm not defined: = reads per million

L. 319: Meydan et al. [19]…..; this Ref. # can be deleted in L. 322

L. 358: move introduction of N-terminomics here from L. 364

Abbreviations not found in Abbreviation list or defined elsewhere: cas = CRISPR-associated;  aTISS; TIGR Annotation Engine, Prokka, DNABERT, Evo 2; GeneLM; GeneMark; Glimmer; TITER; DeepGSR;

References – inconsistencies in title capitalizations; aren’t abbreviations of Journal names preferable

L. 748: remove first “doi.”

L. 712 “volume”—needs article # or Pp.

Other Refs. should be carefully checked for errors.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this review article authors analyzed Technical advance, Challenges, and Functional Insights of bacterial N-Terminal proteoforms. This research article is very interesting to read and is suitable for publication after minor revision.

The work should be hypothesis driven, please do required changes in the introduction section.

Introduction is sufficient to explain the background of this work, but recent references missing (Line 35 to 71).

No clear methodology provided. Methodology may be useful to the readers.

Line 108: Write, “S. Typhimurium”, as “S. typhimurium”. Please use italics appropriately. Please correct Line 635. Please check throughout the manuscript.

How this work is novel than previously published reviews in this subject line?

There is no clear discussion, please include reference appropriately.

Conclusion is not convincing. Please rewrite carefully.

Line 547-555: Please include recent reference

Line 595-605: Include reference

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The review "Advances in Decoding Bacterial N-Terminal Proteoforms: Technologies, Challenges, and Functional Insights" discusses technological advances in the detection of N-terminal proteoforms and evaluates strategies for the functional characterization of N-terminal proteoforms. The manuscript is well structured, has high-quality illustrations, and covers 172 references, of which only 37 are published between 2020 and 2026. The corresponding author is not only an internationally recognized expert in this field but also one of the most highly cited scientists. It is likely that this review was commissioned for MDPI Publishing House. However, a short list of recommendations for authors is provided.

Recommendations:

1. The manuscript lacks a Conclusion or Summary section. Such a section is necessary.
2. It is recommended to revise the cited literature list to increase the proportion of more recent literature. The optimal option would be to replace literature from the last century with literature published between 2020 and 2026, which will contribute to the topic's relevance.
3. Given the addition of a Conclusions or Summary section, it is recommended to revise the abstract to formulate the author's scientific hypothesis and differentiate it from other reviews on this topic.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf