Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

  1. Nicolas Kint
  2. Thomas Dubois
  3. Patrick H. Viollier

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Abstract

Glycosylation of surface structures diversifies cells chemically and physically. Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages. We investigated a new class of FlmG protein glycosyltransferases that modify flagellin, the structural subunit of the flagellar filament. Functional insulation of orthologous Pse and Leg biosynthesis pathways accounted for the flagellin glycosylation specificity and motility conferred by the cognate FlmG in the α-proteobacteria Caulobacter crescentus and Brevundimonas subvibrioides , respectively. Exploiting these functions, we conducted genetic glyco-profiling to classify Pse or Leg biosynthesis pathways and we used heterologous reconstitution experiments to unearth a signature determinant of Leg biosynthesis in eubacteria and archaea. These findings and our chimeric FlmG analyses reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse and that uses specialized flagellin-binding domain to identify the substrate.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

    Major comments:

    The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.

    1.- In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?

    Response: The Caulobacter ΔpseI mutant accumulates low levels of flagellin in the supernatant. TEM analysis reveals that the flagellar filament is not assembled and only the hook structure is visible (PMID: 33108275). Brevundimonas subvibrioides ΔlegI or ΔflmG cells feature a shorter filament compared to WT by TEM. In all these analyses, TEM was performed on cells grown in broth to exponential growth phase as detailed in the Experimental procedures section. These mutant cells do not swim when analyzed by phase contrast microscopy. While is not known if swimming on semi-solid medium would further destabilize the flagellar structures seen in liquid cultures by TEM, there is more residual motility in B. subvibrioides mutants that make a short filament compared to C. crescentus mutants that lack the flagellar filament. Thus, our analyses point to a positive correlation between the residual motility and residual filament length when comparing the B. subvibrioides and C. crescentus mutants.

    2.- In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches. Thanks. Corrected. 3.- In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.

    We thank the reviewer for raising this point. We have now clarified this question in the updated Experimental procedures section. Our immunoblots harbor the same number of cells harvested in exponential phase (OD=0.4). One mL of cells was harvested from cultures by centrifugation at full speed. The supernatant that was used for the immunodetection corresponds to the supernatant after the centrifugation. The supernatant fraction contains flagella that have been shed during the cell cycle at the swarmer cell to stalked cell (G1-S) transition of C. crescentus and B. subvibrioides.

    Thus, it is clear that the majority of flagellins detected by immunoblotting are in fact cell associated and specifically the intracellular flagellins. The evidence for this is that the levels are comparable between WT and ΔflmG mutant cells, even though the latter has shorter or no flagellar filaments. Moreover, while C. crescentus cells are not constantly flagellated during the cell cycle, flagellins are detectable on cell-associated samples by immunoblotting even when cells do not yet or no longer have a flagellar filament. Based on these two points, we conclude that the total flagellin levels associated with cells do not reflect the levels of flagellin assembled into a flagellar filament, but rather the flagellin bulk present in the cytoplasm.

    Consistent with this view, we previously reported that C. crescentus ΔpseI cells have the same amount of flagellins in cell lysates compared to the WT strain (PMID: 33108275), even though the mutant cells lack a flagellar filament. Thus, the results obtained here are consistent with previous observations and indicate that B. subvibrioides flagellin glycosylation mutants also still produce comparable amounts of flagellins intracellularly like the WT strain, despite the absence of flagellin glycosylation and inefficient assembly into a flagellar filament.

    Concerning the potential role of LegI and FlmG in flagellin stability after export, we were referring to protein stability (half-life), not filament stability. Glycosylation may impact the half-life of extracellular flagellins since glycosylation can protect from proteolytic degradation of proteins, possibly in this case by different proteases that may accumulate in the supernatant. Thus, non-glycosylated flagellins could be more easily degraded by extracellular proteases once they are exported, ultimately resulting in a lower amount in the supernatant.

    Addressing the final question about the specificity of the anti-FljKCc antiserum: we used this anti-serum because it detects the B. subvibrioides flagellins owing to the high sequence similarity between B. subvibrioides flagellins and C. crescentus flagellins. We previously showed that the anti-FljKCc anti-serum detects all six flagellins from C. crescentus, as determined by individually expressing each flagellin in a strain deleted for all six flagellin genes (Δfljx6) (PMID: 33108275). FljKCc (against which the antibody was raised) is 65% similar to the most distant C. crescentus flagellin, FljJ. As the similarity of FljKCc to the three B. subvibrioides flagellins ranges from 74% -67% sequence similarity, they should be even better recognized by the anti- FljKCc antibody than C. crescentus FljJ. However, on immunoblots we cannot attribute the signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE and therefore all flagellins might reside in the same immunoblot band. However, we can clearly demonstrate that the immunoblot band corresponds to flagellins: a B. subvibrioides ΔflaF mutant (see below) that we constructed revealed that the flagellin signal is lost, as is the case for a C. crescentus ΔflaF mutant (PMID: 33113346). In the case of C. crescentus, the FlaF secretion chaperone is required for flagellin translation (synthesis) and we suspect that this also the case for B. subvibrioides FlaF. This experiment provides additional evidence that the B. subvibrioides flagellins are recognized by the anti-FljK (C. crescentus) anti-serum.

    4.- Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background. The answer to the first question is “No” or “very inefficiently” as determined from immunoblot analyses of *B. subvibrioides ΔflmG *cells expressing the chimeric FlmG_Cc-Bs protein that we now show in Fig S2B.

    Expression of the different FlmG (FlmG_Cc, FlmG_Bs, FlmG_Cc-Bs) in C. crescentus cells producing Pse or Leg revealed that FlmG_Bs does not support efficient flagellin modification with Pse in C. crescentus, likely because FlmG_Bs interacts poorly with the C. crescentus flagellins. By using the FlmG_Cc-Bs chimera we hoped to overcome this interaction problem with the C. crescentus flagellins (because the FlmG chimera harbors the C. crescentus TPR to bind the C. crescentus flagellins), however glycosyltransfer still does not occur efficiently because the GT domain from FlmG_Bs does not function with Pse. However, FlmG_Cc-Bs can modify the C. crescentus flagellins once C. crescentus is genetically modified to produce CMP-Leg (instead of CMP-Pse). This confirms that the FlmG TPR from C. crescentus is important for flagellin modification through the FlmG/flagellin interaction and that GT_B type glycosyltransferase only transfers Leg. In addition, we have now added as Fig S2B an immunoblot and as Fig S2C a motility test of B. subvibrioides ΔflmG cells expressing the FlmG_Cc-Bs chimeric protein in which we only observed little modification of B. subvibrioides flagellins and a poor motility, respectively. We extended our discussion of these results.

    5.- Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.

    To investigate why the C. crescentus cells that produce Leg and express the chimeric FlmGCc-Bs glycosyltransferase are non-motile (Figure S5B) despite flagellin modification (by immunoblotting, Figure 7C), we employed two strategies. First, we performed immunoblot analyses on the supernatant fraction from these cells to determine if flagellins accumulate extracellularly. As now showed in Figure S5A, only low amounts of C. crescentus flagellins modified by Leg are present in the SN fraction. Second, we conducted TEM analyses of cells grown to exponential growth phase in broth. As shown in Figure S5C, the C. crescentus cells producing Leg and expressing FlmG_Cc-Bs glycosyltransferase harbor a shorter flagellum compared to those expressing the FlmG_Cc in which C. crescentus flagellins are modified by Pse. Altogether these results explain why these cells are non-motile both on soft agar plate and in liquid.

    Minor comments: 1.- Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...." 2.- Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend. 3.- Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)." 4.- Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.

    5.- Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence. 6.- Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......." 7.- In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.

    8.- Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.

    Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....." 10.- Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..." 11.- Introduce a line break between line 503 and 504. 12.- Page 14 line 543, please change "XbaI" to "XbaI" Thanks for the careful editing. We changed the text as suggested by the reviewer. We also added a scheme showing the genetic organization of the genes involved in Leg production and present as Figure 1B. When this study was initiated, the pMT335 plasmid with a Pvan promoter was used before we switched to using the pSRK plasmid with Plac promoter for better induction. Note that the results with Pvan or Plac are comparable regarding the PseI synthases interchangeability. Color code is now homogenous through the manuscript.

    Reviewer #1 (Significance (Required)):

    This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species. Major comments: The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium.

    We have now added the glycopeptide analyses as requested. They are described in the last experimental section and confirm our results.

    The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.

    *The motility pictures are representative of three independent experiments and the immunoblots are representative of at least two independent experiments. This has now been mentioned in the Experimental procedures. The raw unprocessed blots have now been added as supporting info. *

    Minor comments: There are a few textual errors that the authors should fix: -page 2, line 70: change "used" to "use" -page 11, line 407: add the word "are" after Pse On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected. We corrected the text as the reviewer suggested. We changed the sentence in the introduction to be more nuanced. The drawing of the monosaccharide has been improved.

    Reviewer #2 (Significance (Required)):

    The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented. These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.

    Major comments:

    1. Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg". Putative PseI and LegI were proposed based on BlastP analyses in which the protein sequences of interest were aligned to the three experimentally validated synthases from C. jejuni 11168: PseI, LegI, NeuB as well as PseI from C. crescentus, as indicated in Table S1. While, the assignment of the donor sugar is based only on the sequence identity and similarity to LegI or PseI, this assignment corresponds well according to the restoration of the motility of the C. crescentus ΔpseI mutant upon expression of PseI ortholog and B. subvibrioides ΔlegI mutant with heterologous LegI expression.

    It is true that for Shewanella oneidensis the assignment as PseI or LegI is ambiguous, exhibiting nearly identical similarity, but it is quite distinct from NeuB. This actually makes the S. oneidensis synthase a very interesting case to explore the enzymology of its Pse/LegI ortholog, knowing that it has been previously shown that this bacterium glycosylates its flagellins with Pse derivatives (PMID: 24039942). The results from our genetic complementation analysis are however very clear (PseI ortholog) and very consistent with the functional analysis in S. oneidensis.

    Concerning the different derivatives of Pse or Leg: McDonald and Boyd (PMID 32950378) recently published a review giving some examples of Bacteria/Archaea experimentally shown to contain Pse/Leg-derivatives: C. jejuni 11168 modifies its flagellin with 5,7-N-acetyl Pse, Sinorhizobium fredii NGR234 (not used in this study but in our previous work PMID 33113346 and showed to restore the motility of C. crescentus ΔpseI cells) modifies its capsule with 5-acetamido-7-3-hydroxybutyramido-Pse), Treponema denticola modifies its flagellin with 7-(2-metoxy-4,5,6-trihydroxy-hexanoyl-Pse, A. baumannii LAC-4 produces 5,7-N-acetyl-8-epi-Leg to decorate the capsule, Halorubrum sp. PV6 modifies the LPS with N-formylated Leg and L. pneumophila produces 5-acetamidino-Leg.

    The reviewer is right in that we do not know the exact version of Pse or Leg produced in C. crescentus and B. subvibrioides, HOWEVER, the fact that complementation works with the majority of the orthologs of PseI and LegI including many from bacteria that are known to produce modified Pse derivatives for example in Shewanella oneidensis and Treponema denticola, the most likely explanation is that derivatization occurs after the PseI or LegI step, but we concede that the results are also compatible with a promiscuous enzyme that can accept different Pse derivatives or different Leg derivatives.

    Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included. We agree that for many PseI or LegI tested, there is no provided biochemical evidence. HOWEVER, this is not the case for some of them including the PseI, LegI and NeuB from Campylobacter jejuni (PMID 19282391), some A. baumannii strains (α-epi-legionaminic acid for A. baumannii LAC-4 PMID 24690675), Shewanella oneidensis (Pseudaminic acid with methylation PMID 23543712), Legionella pneumophila (Legionaminic acid PMID 18275154) or Halorubrum sp. PV6 (N-formylated legionaminic acid PMID 30245679). Thus, we maintain the two conclusions: the PseI and LegI synthases are generally interchangeable and the complementation assays can enable to identify and assign PseI and LegI function. BLAST2P was used to compare the protein sequences of the tested NeuB-like synthases with NeuB1, LegI (NeuB2) and PseI (NeuB3) from Campylobacter jejuni but also with PseI from C. crescentus. BLOSUM62 matrix was used as well as a word of size 3 for the comparison. We have now added this procedure in the legend of the Table S1.

    It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants? __The number of flagellin subunits that are assembled into a full-length flagellar filament is unknown in C. crescentus and in B. subvibrioides. There are 3 different flagellin genes that are now presented schematically in Figure 1C. No redundancy has been found for LegI or FlmG. It is possible that the B. subvibrioides is better in exporting non-glycosylated flagellin or that the capping proteins can function better with sugar modification or that the filament of B. subvibrioides mutants is less fragile when it is non-glycosylated or that its flagellins “stick” better. It is also possible that short filaments are not actually containing flagellins mounted on the hook but another protein that polymerizes aberrantly in the absence of Leg or FlmG. This remains to be investigated and compared to the situation of Pse and FlmG mutants of C. crescentus. __

    B. subvibrioides possesses an ortholog of the C. crescentus flagellin secretion chaperon FlaF (PMID 33113346). As observed in C. crescentus, FlaF likely has a role in flagellin translation as its inactivation totally prevents flagellins production (see answer to reviewer #1). For C. crescentus, bacterial two hybrid experiments revealed that FlaF can interact with non-glycosylated flagellins in E. coli. Thus, it is strongly possible that FlaF/flagellins interaction is not dependent on the flagellins glycosylation state. In addition, the short flagellum filament observed in B. subvibrioides ΔlegI or ΔflmG mutants argues that at least some flagellins are secreted while not glycosylated.TEM pictures have been performed in liquid medium from exponential growth phase. In this condition, no fragment of flagella was observed in the culture medium by TEM but only small flagella with a hook structure attached. Also, flagella breaks might result in more random length of flagellum.

    Three flagellins are in B. subvibrioides (Bresu_2403 is 59% identical with FljLCc, Bresu_2638 is 57% identical with FljKCc and Bresu_2636 is 62% identical with FljJCc). We now show this genetic organization of the flagellins in Fig. 1C. The three flagellins are all detected by the anti-FljKCc anti serum (see answer and figure to reviewer #1). We cannot attribute the immunoblot signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE. However, the signal often looks like a doublet (as shown in Figure 4B for example) suggesting that at least two flagellins are detected and this doublet is always found to migrate faster in absence of glycosylation that could indicate that all B. subvibrioides flagellins (or at least 2) are modified.

    Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody? Antibodies raised to FljKCc were raised against His6-FljK produced in E. coli (previously published in Ardissone et al, 2020). This serum recognizes the 6 flagellins from C. crescentus (PMID: 33108275). It recognized the three flagellins from B.s. (see answer to reviewer #1).

    It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.

    C. crescentus cells that produce Pse (endogenously) or Leg (reconstituted pathway) and BsFlmG and BsFljK (formerly Figure 5C and now as Figure 7C) are indeed not motile as shown by the motility tests presented in Figure S5B. Motility assays with cells used in the former Figure 5B (now Figure 7B) have also been done and are now presented Figure S4B. These cells are non-motile because BsFljK is not efficiently secreted (or unstable after secretion) as shown on the immunoblot of the supernatant fraction in Figure S4A lower panel. As a result, flagellar filament is not properly assembled as only a short flagellum was observed by TEM in such cells compared to the WT C. crescentus (Figure S4C and S4D).

    Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.

    *__ Immunoblots of the supernatants for Figure 5B (now Figure 7B) have been done and been added (Figure S4A lower panel). BsFljK is barely detected in the supernatant whatever its glycosylation state (with or without Leg). Note that in the supporting info where the raw unprocessed blot used for this panel is shown, a positive control of blotting (C. crescentus Δfljx6 mutant expressing CcFljK from pMT463) has been used. Immunoblots of the supernatant from Figure 5C (now 7C) have been done and been added in figure S5A. The CcFljK modified with Leg is poorly secreted (or unstable after secretion). As a result, these cells only harbor a short flagellum compared to those that are able to modify CcFljK with Pse (Figure S5C).

    HPLC of the acid extracts from flagellins have been performed on purified flagella obtained by ultracentrifugation. As C. crescentus cells expressing BsFlmG and Cc-BsFlmG harbor no or short flagellar filament, the purification by ultracentrifugation is limited. Thus, to further confirm that CcFlmG has transferred Pse and Cc-BsFlmG (and BsFlmG) has transferred Leg on CcFljK (former Figure 5C and now Figure 7C), we performed immunoblots on the cell extracts of C. crescentus ΔflmG ΔpseI cells that cannot produce Pse but able to produce Leg (reconstituted pathway). These experiments, now presented in Figure 7C (lower panel) confirmed that no modification of CcFljK was observed in C. crescentus cells expressing CcFlmG whereas CcFljK is modified in C. crescentus expressing Cc-BsFlmG, confirming that Cc-BsFlmG has transferred Leg (the only NulO produced in this condition).__*

    Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.

    GlmU is a potential bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase that can be part of both Pse and Leg pathway (PMID 19282391). Accordingly, a GlmU ortholog is found in C. crescentus and B. subvibrioides that we showed are producing Pse and Leg, respectively. Thus, GlmU cannot be attributed to a Leg pathway signature. On the other hand, PtmE is barely found in the organisms from which PseI orthologs restore the motility of C. crescentus ΔpseI cells.

    PtmF, PtmA, PgmL and GlmS are proposed to act upstream of the production of GlcN-1-P that is a precursor of both UDP-GlcNAc and GDP-GlcNAc, the precursors of Pse and Leg respectively. In addition, orthologs of these genes are not prevalent in the Leg synthetizing organisms present in Table S2 using BlastP analyses with C. jejuni proteins as templates.PtmE ortholog is found in most of the Leg synthetizing organisms as shown in Table S2 and often genetically linked with other genes coding for proteins involved in Leg production (shown with the asterisk * in table S2). Of note, PtmE is found not only in organisms that modify flagellin(s) with Leg but also in organisms that add Leg on capsule such as A. baumannii LAC-4.

    It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of? The motility pictures are representative of three independent experiments. The immunoblots are representative of at least two independent experiments. This information is now added in the Experimental procedures section.

    Minor comments:

    1. The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.

    2. Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy. Done

    3. Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy Corrected

    4. Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives" corrected

    5. Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.

    6. Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI. Indeed, we modified the sentence.

    7. Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors" Corrected

    8. Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"? Corrected

    9. Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate. Correct. Because we cannot test all enzymes we must keep the statement non-committing.

    “Most (if not all)” refers to the latter case i.e. the modification occurs after PseI synthase. In this context, PseI should do the same reaction, however, there might be some exceptions.

    There is, to our knowledge, no reports showing that modifications occur before the PseI synthase. The glyco-profiling experiments all suggest that modification occurs after Pse production based on our motility readout. It is possible that PseI enzymes that condense a modified precursor would not be functional in our motility assay.

    Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome? We now have modified the sentence to make it clearer.

    At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text. Thanks for raising this issue. We have now tried to address this concern by re-organizing the order of occurrence of the figures. Notably we have now exchanged Figure 5 (on Leg pathway reconstitution and FlmG rewiring) with Figure 7 (on LegB and LegH). We modified the text accordingly. We hope that it makes the manuscript and corresponding figures easier to follow.

    Reviewer #3 (Significance (Required)):

    The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases

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    Referee #3

    Evidence, reproducibility and clarity

    Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented.
    These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.

    Major comments:

    1. Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg".
    2. Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included.
    3. It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants?
    4. Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody?
    5. It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.
    6. Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.
    7. Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.
    8. It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of?

    Minor comments:

    1. The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.
    2. Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy.
    3. Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy
    4. Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives"
    5. Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.
    6. Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI.
    7. Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors"
    8. Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"?
    9. Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate.
    10. Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome?
    11. At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text.

    Significance

    The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species.

    Major comments:

    The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium. The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.

    Minor comments:

    There are a few textual errors that the authors should fix:

    • page 2, line 70: change "used" to "use"
    • page 11, line 407: add the word "are" after Pse
    • On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected.

    Significance

    The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

    Major comments:

    The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.

      • In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?
      • In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches.
      • In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.
      • Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background.
      • Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.

    Minor comments:

      • Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...."
      • Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend.
      • Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)."
      • Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.
      • Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence.
      • Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......."
      • In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.
      • Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.
    9. Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....."
      • Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..."
      • Introduce a line break between line 503 and 504.
      • Page 14 line 543, please change "XbaI" to "XbaI"

    Significance

    This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

  5. Version published to 10.1101/2022.03.25.485807v1 on bioRxiv