Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

  1. Adrià Sogues
  2. Mike Sleutel
  3. Julienne Petit
  4. Daniela Megrian
  5. Nicolas Bayan
  6. Anne Marie Wehenkel
  7. Han Remaut

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Abstract

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or ‘S-layer’, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle ‘core’, a three-helix bundle ‘arm’, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

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  1. Review Commons

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

    Responses

    • The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
    • Though this is not stated in the MS
    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

    Response: We added...

    Referee #2

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript from Sogues et al, the authors investigate the S-layer of Corynebacterium glutamicum, a bacterium extensively used in biotechnological applications, using single-particle cryoEM of purified PS2 S-layer, advanced light microscopy, and bioinformatics. They convincingly demonstrate that the C. glutamicum S-layer consists of hexagonal PS2 arrays and provide the underlying structural basis of this assembly. Furthermore, they nicely analyze the conserved and divergent elements of PS2 across Corynebacteria. Engineering SP2 for its use with the SpyTag-SpyCatcher technology revealed, that SP2 is incorporated in the S-layer at the poles.

    Minor comments:

    • At first it was unclear to me why the authors decided to heterologously express PS2 in C. glutamicum ATCC 13032 if there are other C. glutamicum strains available that naturally harbor an S-layer (e.g. ATCC 13058). It became clear while reading the manuscript but it would help the reader if the authors would clarify and reason their choice of model strain at the beginning of the results section.
    • Figure 3: The font size as well as the panels are very small - please increase the size. In panel d, a schematic showing the location of the funnel would aid in an easy understanding of the figure.
    • Figure 5: Font sizes are very small. Can the authors make it a full-page figure?
    • As this is a back-to-back submission, the manuscript from Isbilir et al. should be cited.
    • Typo: Supplementary Figure 5 is labeled as Supplementary Figure 4 in its title.
    • Typo in several figure legends: um/nm instead uM/nM (micro/nanometer vs. micro/nanomolar).

    Significance

    This is a well-written manuscript, scientifically sound, and the conclusions are convincing. Furthermore, I appreciate that the authors are not overselling their findings.

    The findings in this manuscript are exciting for a specialized audience interested in bacterial cell surfaces/surface appendages and S-layers. On top, as C. glutamicum is widely used in biotechnological applications, the results have clear significance within this field.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Corynebacterium glutamicum is an important organism with industrial applications, and it constitutes a model organism for the study of other Corynebacteriales, which include important pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae. This work provides with a thorough structural and functional characterization of the S-layer structure of C. glutamicum based on solid data acquired through protein engineering, structural biology, and cell microscopy/imaging studies, and phylogenetic analysis. The authors have determined an atomic structural model of the S-layer from C. glutamicum formed by the protein PS2 exhibiting a different degree of conservation between external and mycomembrane facing surfaces; and they show evidence in vivo of how the S-layer assemble at the cell poles in this organism in line with the actinobacterial elongasome. The authors also show that the presence of the S-layer provides resistance to lysozyme, elaborating several hypotheses that may explain this observation, and demonstrate PS2 S-layer as a feasible platform for covalent surface display both in vitro and in vivo.

    The conclusions are well supported by the data provided. When required, experiments have been performed with an adequate number of replicates and their statistical analysis is properly provided. The information provided in the data and methods´ section are sufficient for experimental reproducibility.

    No major comments

    Minor comments. Text and figures are clear but the following aspects/questions should be addressed/clarified:

    • While the authors indicate that "S-layers are two-dimensional monolayered crystals typically composed of a single (glyco)protein ..." (lines 23-24), I haven´t found a reference to whether PS2 is glycosylated or not in the text. If PS2 is glycosylated and its glycosylation sites are known, where would they locate in the glycosylated structure? Would glycosylation affect the size of the pores observed with the recombinant protein?
    • Related to the permeability of lysozyme through the S-layer. Have the authors considered the IP of the lysozyme, which (assuming it is hen white egg lysozyme) it´s > 9. Could the negatively charged PS2 nonspecifically capture/retain at the outward-facing surface? Could glycosylation have something to do with it too?
    • Clarify which AF version was used for predicting the structural model of PS2, AF2 (as described in the main text, lines 160 and 656) or AF3 (supplementary Fig. 2). If it was AF3, the corresponding reference should be updated.
    • Line 205. Replace "Analysis of its surface electrostatics reveals that PS2..." by "Analysis of the electrostatic potential surface of PS2 reveals that..."
    • Figure 2 - panels (e), (g), (i), (j). Cartoons and specially ball-and-stick representations colored in white are very hard to visualize or not visible. Please use a darker color or darken the edges to improve visibility. Labels in panel (g) are very small and difficult to see, please increase their size (panels (i) and (j) seem okay).
    • Line 215. "vivo and recombinant PS2AD" remove "and".
    • Line 1011-12. Replace "...positive in blue to negative in red" by "...positive and negative charges are colored in blue and red, respectively"
    • Supplementary figure 3. Reduce the label size in the why axis (probability)
    • Supplementary figure 5. It is labeled as "4" instead of "5". The view captured in the figure does not allow to visualize the interface clearly. Please consider another orientation or an alternative representation such an open-book view.
    • Supplementary table 1. Replace "BL21(DH3)" by "BL21 (DE3)"
    • Review abbreviations, italics, spaces between number and units... (page 517, C. glutamicum in italic; choose either cryoEM or cryo-EM abbreviation for consistency...)

    Significance

    From my point of view as structural biologist, the present work uncovers key aspects of PS2 S-layer architecture and assemble over the OM of C. glutamicum, together with evidences in vivo of how the S-layer incorporated at cell poles alongside with the bacterial elongasome, and data supporting an implication of the PS2 S-layer in cell envelop stability. These findings constitute an important advance in the structural and functional understanding of S-layer in corynebacteriales, at the time it opens new questions regarding the role of the S-layer in cell integrity and as an interaction interface with external factors. Moreover, the authors present data supporting the feasibility of the PS2 S-layer of C. glutamicum as a protein-based platform to anchor with potential application in bioengineering materials and synthetic biology, and which has the potential to expand the biotechnological/industrial applications of C. glutamicum. Thus, this work has a significant relevance for the scientific community investigating the structure, function and biogenesis of the S-layer and the cell envelop, in particular. But also in a more general outlook, for the scientific community working in the fields of host-pathogen interactions and bioengineering materials.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors investigate the structure of the surface layer, or S-layer, in the Corynebacteriales organism Corynebacterium glutamicum. Studies of the S-layer in Corynebacteriales are lacking and both the function and assembly of the S-layer is unclear in the context of the unique cell envelope of these microbes. In other bacterial systems, the S-layer has been implicated in many critical biological processes. The authors report the ex vivo cryo-EM structure of the S-layer protein PS2 from C. glutamicum, which shows hexameric symmetry with additional trimeric interfaces. The authors show that the C-terminal membrane anchor domain, which is not resolved in the reported structure, is important for lipid binding based on heterologous expression experiments in E. coli. Used the Spytag/Spycatcher tagging system and fluorescence microscopy, the authors determine that S-layer assembly occurs at the cell poles and is likely coordinated the polar growth mechanism of C. glutamicum.

    Major comments:

    1. The description of the different types of symmetry within the PS2 structure was confusing and difficult to correlate with the structure as depicted in Fig. 2. Authors should clarify labeling and coloring in Fig. 2e, g-j.
    2. Investigation of the function of the S-layer as a permeability barrier (Fig. 3e) would be strengthened by testing susceptibility of cells +/- S-layer to different classes of antibiotics or osmotic shock (optional).
    3. Due to the probable importance of the membrane-anchoring domain on S-layer function, can the authors comment on potential predicted structure of the regions of the membrane anchoring domain that was not resolved in their structure? How does this region differ between different Corynebacteriales species (or in S-layer proteins in mycobacterial species) that have different mycomembrane dimensions?
    4. The authors need to clarify if the version of PS2 used in the live cell imaging experiments detailed in Fig. 4d-f are PS2AD or PS2FL. While they show that PS2AD-Spytag is able to self-assemble, it is possible that the dynamics of PS2AD assembly in vivo are very different from PS2FL due to the absence of the membrane-anchoring domain. Comparison of dynamics between these two constructs would also be a nice addition to the paper.
    5. The pulse labeling experiments using Spycatcher would be strengthened by including fluorescent D-amino acids within the same cells to show true co-localization of S-layer assembly and PG synthesis. This could also shed light on the timescale of S-layer assembly in relation to biogenesis of other layers of the cell envelope.

    Minor comments:

    1. line 71: authors should elaborate on terminology "P6 symmetry"
    2. In Fig. 1g, it is not immediately clear that there is lattice formation. Authors should consider including an inset zoomed in box to make this clearer.
    3. line 165, 171, 194: do the authors mean "protomers"? If not, use of the word "promoter" is confusing
    4. citation on line 374 is incorrect. The cited paper focuses on inner membrane proteins that transport mycolic acid. Also, many of the mycomembrane porins, especially in C. glutamicum do not have a beta-barrel structure (see Ziegler et al, JMC, 2008)
    5. Citations detailing polar assembly of other envelope layers would provide additional support for generalized polar assembly of Corynebacteriales cell envelope (arabinogalactan- Marando et al, JACS, 2022) (mycolic acid biosynthesis proteins- Thouvenel et al, Sci Reports, 2023)
    6. Some of the supplementary figures are not referenced in the text

    Significance

    This study is of broad interest to both the Corynebacteriales and S-layer fields. The study is thorough and detailed but could be strengthened by some clarification in how the structure is presented and further discussion of the biological implications of the membrane anchoring domain. There is a long-held interest in understanding how the unique Corynebacteriales cell envelope is assembled, and the work contributes substantially to the field.

    Reviewer expertise: bacterial genetics, bacterial cell envelope, protein transport

  5. Version published to 10.1101/2024.09.05.611363v1 on bioRxiv