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Mechanism of d-alanine transfer to teichoic acids shows how bacteria acylate cell envelope polymers

Nature Microbiology volume 8, pages 1318–1329 (2023)Cite this article

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Abstract

Bacterial cell envelope polymers are often modified with acyl esters that modulate physiology, enhance pathogenesis and provide antibiotic resistance. Here, using the d-alanylation of lipoteichoic acid (Dlt) pathway as a paradigm, we have identified a widespread strategy for how acylation of cell envelope polymers occurs. In this strategy, a membrane-bound O-acyltransferase (MBOAT) protein transfers an acyl group from an intracellular thioester onto the tyrosine of an extracytoplasmic C-terminal hexapeptide motif. This motif shuttles the acyl group to a serine on a separate transferase that moves the cargo to its destination. In the Dlt pathway, here studied in Staphylococcus aureus and Streptococcus thermophilus, the C-terminal ‘acyl shuttle’ motif that forms the crucial pathway intermediate is found on a transmembrane microprotein that holds the MBOAT protein and the other transferase together in a complex. In other systems, found in both Gram-negative and Gram-positive bacteria as well as some archaea, the motif is fused to the MBOAT protein, which interacts directly with the other transferase. The conserved chemistry uncovered here is widely used for acylation throughout the prokaryotic world.

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Fig. 1: dltX is required for LTA d-alanylation in S. aureus.
Fig. 2: DltX interacts with DltB and DltD as part of a three-member membrane protein complex.
Fig. 3: DltX has a highly conserved C-terminal motif that is critical for LTA d-alanylation but not for complex formation.
Fig. 4: The invariant tyrosine of DltX is required for d-alanylation of DltD, and this chemistry is conserved across many bacterial cell envelope polymer acylation systems.

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Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). The following databases were used for this research: UniProt (including UniRef90), InterPro and various NCBI databases such as GenBank. All plasmids and bacterial strains generated during this work are available upon request. Source data are provided with this paper.

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Acknowledgements

We thank S. Moussa and M. Stone for generating plasmids used in this study, and T. Sisley for streaking strains and starting overnight cultures during COVID-related shift work. We thank F. Hammel and S. Early for critical feedback on the manuscript, as well as all members of the Walker Lab for helpful scientific discussions. We thank T. Pang from the Bernhardt and Rudner laboratories for pTP16. We thank R. Tomaino of the Harvard Medical School Taplin Mass Spectrometry Facility for MS analysis, as well as J. Paulo of the Gygi laboratory for help with preliminary MS experiments. Funding for this work was provided by the National Institutes of Health grants P01 AI083214 and U19 AI158028 to S.W. E.D.S. acknowledges support from National Institutes of Health grant T32 GM139775. B.J.S. acknowledges support from the National Science Foundation Graduate Research Fellowship Program under grant nos. DGE 1745303 and DGE 2140743. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

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Authors and Affiliations

  1. Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Bailey J. Schultz, Eric D. Snow & Suzanne Walker

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Contributions

B.J.S. and S.W. conceived the project. B.J.S. and E.D.S. designed and performed experiments and analysed data, with input from S.W. B.J.S. and S.W. wrote the original draft of the manuscript and, along with E.D.S., revised the manuscript.

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Correspondence to Suzanne Walker.

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Extended data

Extended Data Fig. 1 S. aureus strains without dltX cannot survive on tunicamycin but still produce LTAs.

a, Chemical structures of three repeat units of Staphylococcus aureus LTA (left) and one repeat unit of Streptococcus pneumoniae LTA (right)2. d-Alanine esters are shown in blue. b, Full set of controls for the spot titer assay shown in Fig. 1c. S. aureus strains were grown on tryptic soy agar (TSA) plates containing the indicated compounds: dimethyl sulfoxide (DMSO; 1.25 µL per 1.00 mL of TSA), isopropyl-β-d-1-thiogalactopyranoside (IPTG; 1.00 mM), and/or tunicamycin (tunic.; 1.0 µg/mL). Tunic. inhibits WTA biosynthesis, IPTG was used to induce expression of the indicated plasmid-borne cassette, and DMSO was used as a vehicle control. The image of the plate with tunicamycin and IPTG has been reproduced here for clarity. The plasmid harboring dltXABCD is leaky thus resulting in growth of the sixth strain even in the absence of IPTG. c, An α-LTA western blot indicates that all of the strains tested in b and Fig. 1c produce LTAs at roughly equivalent levels to wild-type S. aureus. The strain in Lane 2 (4S5)75 is a mutant that lacks LtaS and contains a suppressor mutation that permits growth in the absence of LTAs. It does not produce any LTAs. The LTAs themselves run as a smear from ~15–37 kDa. d, Spot titer assay results for a partially different set of S. aureus strains. Here, the complementation constructs are under anhydrotetracycline (aTc)-inducible control, and the overall cassettes are genomically integrated. The TSA plates here contained DMSO, tunic., and aTc as indicated, with the concentrations of DMSO and tunic. being the same as for b and that for aTc being 0.4 µM. In these strains, the dltXABCD complementation construct does not appear leaky. e, SDS-PAGE autoradiography of LTAs from S. aureus cells grown in the presence of d-[14C]alanine again shows that dltX is required for LTA d-alanylation. Here, the same set of strains from d was used. f, An α-LTA western blot indicates that all strains tested in d and e produce LTAs at roughly equivalent levels to wild-type S. aureus.

Source data

Extended Data Fig. 2 DltX is a predicted bitopic transmembrane protein with an extracellular C-terminus and is required for DltD to copurify with DltB.

a, (top) The TOPCONS web server66 for consensus-based membrane protein topology prediction was used to predict the topology of S. aureus DltX. The sequence of S. aureus DltX is shown with the predicted localization of each individual residue indicated beneath. (bottom) An AlphaFold240 model of S. aureus DltX generated using ColabFold41. This specific model is from an overall model of DltX with DltD and was selected here because it illustrates the topology of the protein well. We note that in the DltDX model, DltX’s predicted topology is in agreement with the known topology31 of DltD. b, An α-myc western blot indicates that a similar amount of myc-StDltD was present in the solubilized membranes loaded onto the TALON immobilized metal affinity chromatography (IMAC) resin during purifications of StDltB-His10 (and interactors) from E. coli cells expressing StdltB and StdltD, with or without StdltX. A similar amount of myc-StDltD was also present in the flow-through from the resin in the two samples. However, no myc-StDltD could be observed in the IMAC elution fraction from the sample derived from E. coli cells that did not express StdltX.

Source data

Extended Data Fig. 3 The predicted S. aureus DltBDX structure is a high-confidence prediction.

a, (top) The top-ranked AlphaFold2 model of the S. aureus DltBDX complex, with proteins colored as in Fig. 2c, is a high-confidence model. The predicted local Distance Difference Test (plDDT) is a metric of model confidence, with plDDT values > 90 representing very high confidence, 70–90 representing moderate confidence, 50–70 representing low confidence, and < 50 representing very low confidence. The graph in the upper right shows the plDDT values at each residue for all five models. The numbering on the x-axis here corresponds to the residue number for the concatenated sequence of the complex (that is, the sequence of DltX concatenated N-terminally to the sequence of DltB, and then that overall sequence concatenated N-terminally to the sequence of DltD). The rightmost of the two models underneath the ‘Rank 1’ heading shows a graphical depiction of the plDDT values with a color gradient scale (dark blue representing a plDDT value of 100, and red representing a plDDT value of 0). (bottom) Each of the other four models is highly similar to the top-ranked model. Five models were generated by ColabFold, and the models were ranked by predicted template modeling (pTM) scores. b, The relative positions between the three proteins are high-confidence based on the Predicted Aligned Error (PAE). Here, the value at each (x, y) point in the graphs indicates the ‘expected position error at residue x if the predicted and true structures were aligned on residue y’ (https://alphafold.ebi.ac.uk/faq). Low values (blue) indicate low expected position error. Again, the numbering on the x-axis corresponds to the residue number for the concatenated sequence of the complex. Numbering on the y-axis (not shown) would place ‘0’ at the top of the axis. c, Views of the surface representation of the S. aureus DltBDX AlphaFold2 model.

Extended Data Fig. 4 The C-terminal motif of DltX, and particularly its invariant tyrosine residue, is required for S. aureus growth on tunicamycin.

a, Full set of controls for the spot titer assay shown in Fig. 3b. S. aureus strains were grown on TSA plates containing the indicated compounds: DMSO at 1.25 µL per 1.00 mL of TSA, IPTG at 1.00 mM, anhydrotetracycline (aTc) at 0.4 µM, and tunicamycin at 1.0 µg/mL. Tunic. inhibits wall teichoic acid biosynthesis, IPTG was used to induce expression of the indicated plasmid-borne cassette (here, null refers to a strain with an empty IPTG-inducible cassette in the plasmid), aTc was used to induce expression of the indicated chromosomally integrated cassette, and DMSO was used as a vehicle control for the tunic. The image of the plate with tunic., IPTG, and aTc has been reproduced here for clarity. flag-dltX∆motif encodes for an N-terminally FLAG-tagged DltX variant lacking the last six amino acids. b, Full set of controls for the spot titer assay shown in Fig. 3c. Compounds were used at the same concentrations as above, for the same reasons as above. Here, the bracketed sequences in the superscripts of the aTc-inducible cassettes represent the C-terminal motif sequence of the given DltX variant. Red letters denote changes from the wild-type sequence.

Extended Data Fig. 5 Length alterations at the C-terminus of DltX are not well-tolerated.

a, Full set of controls for the spot titer assay shown in Fig. 3d. S. aureus strains were grown on TSA plates containing the indicated compounds: DMSO at 1.25 µL per 1.00 mL of TSA, IPTG at 1.00 mM, anhydrotetracycline (aTc) at 0.4 µM, and tunicamycin at 1.0 µg/mL. Tunic. inhibits wall teichoic acid biosynthesis, IPTG was used to induce expression of the indicated plasmid-borne cassette (here, null refers to a strain with an empty IPTG-inducible cassette in the plasmid), aTc was used to induce expression of the indicated chromosomally integrated cassette, and DMSO was used as a vehicle control for the tunic. The image of the plate with tunic., IPTG, and aTc has been reproduced here for clarity. Here, the bracketed sequences in the superscripts of the aTc-inducible cassettes represent the C-terminal motif sequence of the given DltX variant. Red letters denote changes from the wild-type sequence, and a red hyphen denotes the deletion of a residue. b, A spot titer assay shows that, of all the DltX mutants we have tested that do not allow for growth of a dltX-null strain on tunicamycin when expressed at low levels (from a chromosomally integrated aTc-inducible cassette), only the DltX mutant with an alanine added onto the C-terminus can complement when expressed at high levels (from an IPTG-inducible cassette on a medium copy number plasmid). Here, dltX* indicates that the plasmid encodes for a variant of DltX with the C-terminal sequence indicated in the ‘DltX C-term.’ column. Again, red letters denote changes from the wild-type sequence, and a red hyphen denotes the deletion of a residue. Both dltXwt (wild-type) and all dltX* here encode an N-terminal FLAG tag.

Extended Data Fig. 6 The S. aureus DltBX and DltDX predicted structures are high-confidence predictions.

a, The top-ranked AlphaFold2 model of the S. aureus DltBX complex is a high-confidence model. The predicted local Distance Difference Test (plDDT) is a metric of model confidence, with plDDT values > 90 representing very high confidence, 70–90 representing moderate confidence, 50–70 representing low confidence, and < 50 representing very low confidence. The graph in the upper right shows the plDDT values at each residue for five models. The numbering on the x-axis corresponds to the residue number for the concatenated sequence of the complex (that is, the sequence of DltB concatenated N-terminally to the sequence of DltX). The rightmost of the two full model images depicts the plDDT values with a color gradient scale (dark blue representing a plDDT value of 100, and red representing a value of 0). The zoom-in image shows how the DltX C-terminal motif fits into the DltB tunnel. b, The relative positions between the proteins are high-confidence based on the Predicted Aligned Error (PAE). Here, the value at each (x, y) point in the graphs indicates the ‘expected position error at residue x if the predicted and true structures were aligned on residue y’ (https://alphafold.ebi.ac.uk/faq). Low values (blue) indicate low expected position error. Again, the numbering on the x-axis corresponds to the residue number for the concatenated sequence of the complex. Numbering on the y-axis (not shown) would place ‘0’ at the top of the axis. c, The top-ranked AlphaFold2 model of the S. aureus DltDX complex is a high-confidence model. The rightmost of the two full model images depicts the plDDT values with a color gradient scale matching that found in a. The zoom-in image shows how the DltX C-terminal motif fits into the DltD active site region. The graph in the lower right shows the plDDT values at each residue for five models. The numbering on the x-axis here parallels that in a. d, The relative positions between the proteins are high-confidence based on the PAE. The numbering on the x- and y-axes parallels the numbering in b.

Extended Data Fig. 7 DltB, DltD, and DltX levels are comparable between samples in the DltD d-alanylation in vitro reconstitution assay, and active forms of all three proteins plus DltA and DltC allow for rapid d-alanylation of DltD.

a, A Coomassie-stained SDS-PAGE gel from an in vitro reconstitution experiment run in parallel to the one shown in Fig. 4b but with cold d-alanine rather than d-[14C]alanine. Lane 4 contains all five active proteins, whereas Lanes 3, 5, and 6 are missing the indicated protein(s). Lanes 7–9 contain four active proteins and the indicated mutant. b, A Coomassie-stained SDS-PAGE gel (run using a Tris-glycine gel rather than a Bis-Tris gel as in a) of purified StDltBDX complex, specifically containing wild-type DltB, inactive DltX (Y56F), and wild-type DltD, provided as a reference for comparison to the samples in a. This suggests the impurity below DltB in a may be a degradation product. c, SDS-PAGE autoradiograph from a time course of the in vitro reconstitution with DltA, DltC, and DltBDX. For the ‘10 sec.’ time point, the DltBDX complex was added to the DltC charging reaction, the sample was mixed by pipetting up and down several times, and then immediately 4x XT sample buffer was added and mixed followed by flash freezing of the sample in liquid nitrogen. For all other time points, after the DltBDX complex was added to the DltC charging reaction, samples were incubated at 30 °C for the indicated amount of time before addition of SDS-PAGE loading buffer and immediate flash freezing in liquid nitrogen.

Source data

Extended Data Fig. 8 DltB, DltD, and DltX levels are comparable between samples in the DltX d-alanylation in vitro reconstitution assay, and d-alanylation of DltXY56K is position-specific.

a, A Coomassie-stained SDS-PAGE gel from an in vitro reconstitution experiment run in parallel to the one shown in Fig. 4c but with cold d-alanine rather than d-[14C]alanine. Lanes 2 and 3 contain wild-type (wt) 1× or 3xFLAG-DltX, while Lanes 4 and 5 contain 1x or 3xFLAG-DltXY56K. b, A Coomassie-stained SDS-PAGE gel (run using a Tris-glycine gel rather than a Bis-Tris gel as in a) of purified StDltBDX complex, specifically containing wild-type DltB, DltXN57K, and wild-type DltD, provided as a reference for comparison to the samples in a. This suggests that the background smearing in the Coomassie-stained gels in a and c (right) is a result of the high-pH reaction conditions combined with the specific gel type used (see Supplementary Fig. 2a for further evidence of this). Additionally, the impurity below DltB in a and c (right) may be a degradation product. We note that the mobility of 1xFLAG-DltX in the Bis-Tris gel here is different from that in the Tris-glycine gel in a, but the bands were confirmed by western blot (See Supplementary Figs. 2b-c). c, (left) SDS-PAGE autoradiography of in vitro reconstitution results shows that the DltXY56K mutant is d-alanylated in a position-specific manner. All lanes contain wild-type DltA, DltC, and DltBDX complex, with the exception that the DltX in the complex is the variant with the C-terminal sequence indicated by the key. These reconstitutions were run alongside those shown in Fig. 4c, and the samples were run on the same gel and thus imaged together. (right) A Coomassie-stained SDS-PAGE gel from an in vitro reconstitution run in parallel to the one shown in c (left) but with cold d-alanine rather than d-[14C]alanine. Again, all lanes contain wild-type DltA, DltC, and DltBDX complex, with the exception that the DltX in the complex is the variant with the C-terminal sequence indicated by the key. In the key, blue coloring indicates the lysine, and red coloring indicates the tyrosine-to-phenylalanine swap.

Source data

Extended Data Fig. 9 A C-terminal motif reminiscent of DltX’s is found at the C-termini of MBOAT proteins that partner with DltD-like proteins in various prokaryotic cell envelope polymer acylation pathways.

a, Graphical depictions illustrate the genomic colocalization of genes encoding MBOAT proteins (green) and DltD-like proteins (blue) in bacterial cell envelope polymer acylation systems, as well as a system of unknown function found in some archaea. The chemical structures show a portion of the relevant polymer for each system, with the modification (acetylation in each of these cases), in blue54,57,76. The lower right depicts a potential polymer acylation system of unknown function found in the archaeal species Nitrosopumilus adriaticus. We have designated the genes mbt1 for MBOAT1 and mbtp1 for MBOAT1 Partner. b, AlphaFold2 models of the two-protein complexes encoded by the genes from a. The models are all shown cytosolic side-down. c, Sequence logo generated by EVcouplings70 with N. gonorrhoeae PatA as the query sequence showing the conservation of the final 51 amino acid positions of an alignment of bacterial cell envelope acylation-associated MBOAT proteins (as well as similar MBOAT proteins of unknown function), with the overall consensus sequence and corresponding NgPatA sequence shown underneath. The height of each individual letter represents the degree of conservation of that specific amino acid at the given position. The final transmembrane (TM) helix indicated is a prediction based on AlphaFold. d, Active sites of the MBOAT proteins of three of the systems shown in this figure (the corresponding active site for NgPatA is found in Fig. 4f) from AlphaFold2 models of the MBOAT proteins alone. These images are all shown with an ‘aerial’ view of the active site (looking toward the cytosol from the extracytoplasmic region). The catalytic histidine known to be required for all studied MBOAT proteins is shown in cyan, and the invariant tyrosine present in this class of MBOAT proteins is shown in purple.

Extended Data Fig. 10 The MBOAT proteins in non-Dlt pathway cell envelope acylation systems have additional C-terminal helices that allow the C-terminal motif of these proteins to nestle into the active site.

Alignments of predicted structures of MBOAT proteins from bacterial cell envelope polymer acetylation systems (and a system of unknown function in archaea) with the predicted structure of the DltBX complex show that the non-DltB MBOAT proteins likely share a similar architecture to DltB but with a C-terminal extension of several additional helices. All of the predicted structures here were generated using the ColabFold implementation of AlphaFold2 and aligned using the PyMOL ‘super’ command.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–6, Tables 19–22 and references.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–18.

Supplementary Data 1

All ColabFold output files for the AlphaFold predictions generated in this work.

Source data

Source Data Fig. 1

Unprocessed autoradiograph.

Source Data Fig. 2

Unprocessed gels and western blot.

Source Data Fig. 3

Unprocessed gel.

Source Data Fig. 4

Unprocessed autoradiographs.

Source Data Extended Data Fig. 1

Unprocessed western blots and autoradiograph.

Source Data Extended Data Fig. 2

Unprocessed western blot.

Source Data Extended Data Fig. 7

Unprocessed gels and autoradiograph.

Source Data Extended Data Fig. 8

Unprocessed gels and autoradiograph.

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Schultz, B.J., Snow, E.D. & Walker, S. Mechanism of d-alanine transfer to teichoic acids shows how bacteria acylate cell envelope polymers. Nat Microbiol 8, 1318–1329 (2023). https://doi.org/10.1038/s41564-023-01411-0

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