Hierarchical cross-linking of a bacterial spore coat Hub protein

Reviewer #1 (Public review):

This is an important article, which represents the culmination of 25 years of research on the spore coat protein, SafA. Reading this paper is not necessarily easy because it requires time, patience, and attention to detail, but it is truly rewarding. The attentive reader will certainly appreciate the description of a biochemical tour de force, providing convincing experimental evidence for every aspect of a step-by-step inner coat assembly model. It was previously known that SafA was a coat morphogenetic protein responsible for the assembly of the inner layer of the spore coat in Bacillus subtilis, and SafA was already viewed as a hub that directly or indirectly recruited several dozens of coat proteins to the spore envelope. It was also known that there were isoforms of SafA (the most important being the C30 form), and SafA was a substrate of Tgl, a transglutaminase involved in crosslinking some of the coat proteins, especially those found in the inner coat. Several studies have combined genetics and various types of microscopy approaches, including fluorescence microscopy, to decipher the mechanism of coat assembly, but the current study brings top-notch biochemistry into the picture and, therefore, is able to go much further into the molecular characterization of this important mechanism. It should be noted that spore coat assembly is a notoriously difficult process to study biochemically. It was also suspected to be a complex mechanism, because coat assembly is a protracted process involving at least 80 different proteins, whose production is controlled both temporally and spatially, but the current paper manages to connect specific chemical reactions to well-known stages of spore formation. The authors did so by generating several constructs with specific substitutions of Cys and Lys residues, interfering with the completion of disulfide bond formation and crosslinking events, thus determining the order of events and the structural consequences when one of these steps is impaired. Importantly, their conclusions are consistent with previous work. In the updated model, self-assembly of SafA is the first step, promoted by disulfide bond formation between C30 complexes. This is followed by recruitment of inner coat proteins and, finally, transglutamination to stabilize the scaffold structure (referred to as a "spotwelding activity".

The work is extremely thorough. I did not identify any weaknesses and could not think of any experiment that would have been omitted.

Reviewer #2 (Public review):

Summary:

The authors assemble a variety of information from biochemical experiments on oligomeric and higher-order assembly of the spore coat protein SafA, which functions as a hub in spore coat development. Together, the data indicate a robust process of assembly, guided initially by an organized process of disulfide bond formation and ultimately leading to cross-linking by the enzyme Tgl. Interestingly, neither process is strictly necessary for the formation of highly assembled oligomeric forms of SafA, but instead, these processes are mutually supportive in creating a strong, intercrosslinked assembly. Given this lead-up, it is somewhat disappointing to find that the cross-linking defective SafA mutants do not exhibit any obvious defects in sporulation in vivo, and one is left with the conclusion that this stage of spore coat assembly is accomplished by multiple independent co-occurring activities. The information is sufficient to support a detailed model for SafA assembly, which is significant in that it helps to explain the process of building a critically important hub-scaffold for spore coat development.

Strengths:

The main body of experiments supports a detailed model for the assembly of SafA monomers into spore coat superstructures. This is interesting because it shows how a protein can be used as both a scaffold and a hub in contributing to the assembly of a super-resilient biological material.

Weaknesses:

(1) The weak sporulation phenotype of the crosslinking mutants diminishes the significance of the mechanism that is described.

(2) The narrative flow of the originally submitted manuscript could be improved by removing some unnecessary and confusing figures on peripheral subjects and rearranging some of the latter figures to arrive at a conclusion that focuses more on SafA assembly.

(3) The original manuscript appears to have a labeling error in the supplementary figures, but a correctly labeled version of the figures would not support one of the manuscript's claims.

Reviewer #3 (Public review):

The manuscript by Amara et al. provides novel mechanistic insight into how SafA, a spore coat morphogenetic protein, self-assembles and is later crosslinked by the Tgl transglutaminase during spore coat assembly. Through rigorous, carefully executed biochemical analyses of SafA's oligomerization and crosslinking states, the authors demonstrate that SafA forms dimers that promote disulfide bond formation between two cysteine pairs found in its C30 region; this disulfide bond-mediated crosslinking promotes, but is not essential for, Tgl-mediated crosslinking of lysine residues within SafA. Specifically, one pair in its N-terminal C30 region promotes the formation of higher-order oligomers, while the second pair in its C-terminus C30 region promotes its ability to form a tetramer. Mutation of both cysteine pairs prevents higher-order SafA structures and reduces the efficiency of Tgl-mediated crosslinking via lysines in close proximity to the cysteines. They further show that disulfide bond formation promotes, but is not essential for, SafA to self-assemble into structures ~1200 kDa via SAXS analyses and kinetic analyses of Tgl-mediated crosslinking of purified SafA in vitro.

Major Comments:

(1) While the authors' detailed and thorough biochemical analyses advance our understanding of how SafA forms higher-order structures in the presence and absence of Tgl, they could broaden the significance of their findings with additional functional analyses of their mutants in B. subtilis. Figure 8 shows that loss of Tgl and SafA disulfide bond formation renders SafA more extractable (presumably leading to a less resilient spore coat), and FRAP analyses indicate that SafA in ∆tgl sporulating cells is more mobile than in its lysine crosslinked form. Some ideas that the authors could test to try and identify additional functions for the Cys and Lys residues in SafA:
- Analyze the Cys mutants in the FRAP assay?
- Does loss of SafA-mediated crosslinking via the Cys and/or Lys mutations affect its localization to the forespore or the recruitment of its client proteins like GerQ?
- Have the authors tested higher concentrations of lysozyme? Or chloroform?

(2) While the authors show in supplementary data that the safA point mutants they generated do not affect spore germination in the single condition tested, the Rudner group previously showed that SafA plays a role in spore germination by affecting CwlJ localization to the forespore. Perhaps the authors might see a more significant phenotype on spore germination with their Cys and Lys mutants if they tried to complement a ∆safA∆sleB double mutant with mutant safA constructs? For the germination assays, it was unclear to me whether the authors used heat activation prior to inducing spore germination.

(3) Have the authors looked at whether the Cys or Lys mutations affect the sensitivity of spores to oxidative insults, especially since the Cys residues might temper the effects of oxidizing agents?

(4) Did the authors test the effect of single Cys mutations on disulfide bond formation, since intermolecular disulfide bond formation might still be possible even if one of the Cys residues has been changed?

(5) Finally, I was unsure how many times each experiment was replicated and how many experiments had been conducted in total.