High-throughput Automated Muropeptide Analysis (HAMA) Reveals Peptidoglycan Composition of Gut Microbial Cell Walls

Reviewer #1 (Public Review):

The paper from Hsu and co-workers describes a new automated method for analyzing the cell wall peptidoglycan composition of bacteria using liquid chromatography and mass spectrometry (LC/MS) combined with newly developed analysis software. The work has great potential for determining the composition of bacterial cell walls from diverse bacteria in high-throughput, allowing new connections between cell wall structure and other important biological functions like cell morphology or host-microbe interactions to be discovered. In general, I find the paper to be well written and the methodology described to be useful for the field. However, there are areas where the details of the workflow could be clarified. I also think the claims connecting cell wall structure and stiffness of the cell surface are relatively weak. The text for this topic would benefit from a more thorough discussion of the weak points of the argument and a toning down of the conclusions drawn to make them more realistic.

Specific points:

1. It was unclear to me from reading the paper whether or not prior knowledge of the peptidoglycan structure of an organism is required to build the "DBuilder" database for muropeptides. Based on the text as written, I was left wondering whether bacterial samples of unknown cell wall composition could be analyzed with the methods described, or whether some preliminary characterization of the composition is needed before the high-throughput analysis can be performed. The paper would be significantly improved if this point were explicitly addressed in the main text.

2. The potential connection between the structure of different cell walls from bifidobacteria and cell stiffness is pretty weak. The cells analyzed are from different strains such that there are many possible reasons for the change in physical measurements made by AFM. I think this point needs to be explicitly addressed in the main text. Given the many possible explanations for the observed measurement differences (lines 445-448, for example), the authors could remove this portion of the paper entirely. Conclusions relating cell wall composition to stiffness would be best drawn from a single strain of bacteria genetically modified to have an altered content of 3-3 crosslinks.

Reviewer #2 (Public Review):

The authors introduce "HAMA", a new automated pipeline for architectural analysis of the bacterial cell wall. Using MS/MS fragmentation and a computational pipeline, they validate the approach using well-characterized model organisms and then apply the platform to elucidate the PG architecture of several members of the human gut microbiota. They discover differences in the length of peptide crossbridges between two species of the genus Bifidobacterium and then show that these species also differ in cell envelope stiffness, resulting in the conclusion that crossbridge length determines stiffness.

The pipeline is solid and revealing the poorly characterized PG architecture of the human gut microbiota is worthwhile and significant. However, it is unclear if or how their pipeline is superior to other existing techniques - PG architecture analysis is routinely done by many other labs; the only difference here seems to be that the authors chose gut microbes to interrogate.

I do not agree with their conclusions about the correlation between crossbridge length and cell envelope stiffness. These experiments are done on two different species of bacteria and their experimental setup therefore does not allow them to isolate crossbridge length as the only differential property that can influence stiffness. These two species likely also differ in other ways that could modulate stiffness, e.g. turgor pressure, overall PG architecture (not just crossbridge length), membrane properties, teichoic acid composition etc.