Main

The MBOAT superfamily comprises more than 7,000 proteins (see http://pfam.xfam.org/family/MBOAT). These proteins perform divergent functions with distinct substrate preferences, although many use acyl-coenzyme A (acyl-CoA) as the acyl-group donor (Extended Data Fig. 1). Among bacterial MBOATs, DltB is essential for the d-alanylation of cell-wall teichoic acids11,12,13,14,15,16, which are important for the growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. To understand the molecular mechanisms of MBOAT proteins, we have determined the crystal structure of full-length DltB from Streptococcus thermophilus at 3.3 Å resolution (Fig. 1, Extended Data Figs. 2, 3, Extended Data Table 1). DltB contains 415 residues arranged into 17 helices, and both the N and the C termini are located in the extracellular space (Fig. 1a). The helices are located mostly within the lipid bilayer, with the exception of the short N- and C-terminal helices. Among them, 11 transmembrane helices form an external ring-shaped ridge, and shield a central basin that is thinner than the lipid bilayer (Fig. 1, Extended Data Fig. 4). The thin central area results from an intracellular concave surface and a more pronounced extracellular structural funnel (Fig. 1d). Because they are more conserved than the peripheral-ring helices among MBOAT proteins and are probably involved in catalysis (see below), we refer to the structural components in this thin central area as the MBOAT central core. The 3D structure of DltB can be approximately divided into three parts: the N-terminal helical ridge (N-ridge), the central core and the C-terminal helical ridge (C-ridge) (Extended Data Fig. 4). A Dali search using our DltB structure did not find any protein with a similar fold.

Fig. 1: Overall structure of DltB and its conserved extracellular funnel.
figure 1

a, The DltB crystal structure is shown in three orientations with rainbow colours: bottom, front and back (from left to right). b, Cartoon of the transmembrane topology of DltB. DltB contains a ring of 11 peripheral transmembrane helices, which shield a central thin layer (the structural core) highlighted by two red dashed circles. c, The electrostatic surface of DltB. d, A cut-away surface illustration showing the outward funnel connected with the cytosolic side through a tunnel. The histidine residue that is completely conserved among MBOATs (His336) is located at the bottom of the funnel. e, Conservation of the extracellular DltB funnel surface. The surface conservation pattern was generated on the basis of sequence alignment shown in Extended Data Fig. 5. f, Top view of DltB showing the location of His336 and the other three DltB residues (Ser165, Ala209 and Phe250) that, when altered, were found to desensitize S. aureus to the inhibition of LTA d-alanylation by m-AMSA.

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The extracellular side of DltB forms a structural funnel, which extends into the middle of the lipid bilayer (Fig. 1d). The surface inside the funnel is formed by residues from several transmembrane helices and loops. Notably, in sharp contrast to the low conservation of residues forming the outer-ridge surfaces, these inner residues are highly conserved among DltB proteins (Fig. 1e, Extended Data Fig. 5). Previous studies have shown that a histidine residue strictly conserved in all confirmed MBOAT proteins is probably involved in catalysis. Mutation of the corresponding histidine residue in all tested MBOATs—porcupine (PORCN), hedgehog acyltransferase (HHAT), ghrelin O-acyltransferase (GOAT), diacylglycerol acyltransferase 1 (DGAT1) and acyl-coenzyme A:cholesterol acyltransferase (ACAT)—either abolished or substantially reduced the acyltransferase activities of the enzymes17,18,19,20,21,22. In our DltB structure, this histidine residue (His336, the last residue of helix H14) is located at the bottom of the extracellular funnel (Fig. 1d, f). Another highly conserved histidine residue (His289) in the MBOAT superfamily1 is also located at the bottom of this funnel and is spatially close to His336 (Extended Data Fig. 3). Our crystal structure and the structural conservation strongly suggest that this extracellular funnel is important for the activity of DltB.

Four Staphylococcus aureus DltB mutations—corresponding to S. thermophilus DltB mutants S165T, A209D, F250L and F250I—have been identified as resistant to the DltB inhibitors m-AMSA (amsacrine) and o-AMSA14. Ser165, Ala209 and Phe250 are spatially located at the surface of the funnel, with Ser165 and Phe250 sitting near the bottom of the funnel and close to His336 (Fig. 1f). We predict that m-AMSA and o-AMSA bind in this DltB funnel, and that the abovementioned four mutations may abolish inhibitor binding. We speculate that this funnel may be involved in extracellular teichoic acid substrate binding or have other key roles in catalysis. Given the biological importance of DltB14 and the marked conservation of the extracellular funnel surface of DltB, inhibitors of DltB that bind to this funnel may act as wide-spectrum antibiotics against Gram-positive bacteria.

In addition to its role in d-alanylation, DltB also has a role in host–pathogen interactions. A missense mutation (T113K) in S. aureus DltB is sufficient to convert an S. aureus strain from a human-specific pathogen to a rabbit-specific pathogen, without any change in the d-alanylation level of lipoteichoic acid (LTA)23. Notably, Thr113—as well as all ten other S. aureus DltB residues that are associated with a change in host specificity—is located at a non-conserved extracellular apex (Extended Data Figs. 4d, 5). This unusual feature strongly suggests that DltB from S. aureus and potentially some other species may interact with one or more unknown host factors using their extracellular ridges.

To serve as the d-alanyl donor to teichoic acid in the Dlt-mediated d-alanylation system, DltC first needs to be modified with the 4′-phosphopantetheine (Ppant) group at Ser35, a modification that can be catalysed by acyl carrier protein synthase (AcpS). The Ppant-modified DltC can be further modified with a d-alanyl group by DltA, through a thioester bond (Fig. 2a). To test whether DltB can directly interact with DltC, we co-expressed His-tagged AcpS and GST-tagged DltC. The purified DltC was uniformly modified by Ppant, as confirmed by mass spectrometry (Extended Data Fig. 2b, c). GST pull-down and size-exclusion chromatography experiments showed that DltC-Ppant and DltB form a tight complex (Fig. 2b). Octet binding analysis showed a Kd of 0.26 µM between DltB and DltC-Ppant (Extended Data Fig. 6). In contrast to the tight DltB–DltC interaction, DltB does not form a detectable complex with DltA or the extracellular domain of DltD, and there is no detectable interaction between DltA and DltC on the cytoplasmic side (data not shown).

Fig. 2: Structural basis of the DltB–DltC-Ppant interaction.
figure 2

a, Dlt proteins responsible for LTA d-alanylation in Gram-positive bacteria. The magenta dots on glycerophosphate units represent d-Ala moieties. b, Direct stable interaction between DltB and DltC-Ppant, and mutagenesis analysis of the DltB–DltC-Ppant interface, as shown by GST pull-down assays. GST pull-down experiments were performed at least twice with similar results. WT, wild type. c, Overall structure of the DltB–DltC-Ppant complex. DltC-Ppant binds to DltB on the cytosolic side, with the phosphate group of Ppant (which is attached through Ser35 of DltC) pointing towards the DltB tunnel. d, The DltB–DltC-Ppant interface. Side chains corresponding to DltB are shown as green sticks, and side chains of DltC are shown in cyan.

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To understand how DltB functions as an MBOAT, we also determined the crystal structure of the DltB–DltC-Ppant complex at 3.15 Å resolution (Fig. 2c). Cytoplasmic DltC contains four helices, with Ppant-bonded Ser35 being the first residue of helix 3 (α3). Residues of DltC α3 and the long loop between α3 and α4 (α3–α4 loop) form the DltB-binding surface. DltC interacts mainly with the C-terminal half of DltB H13 and the N-terminal end of DltB H14. This region is formed by a DltB-specific insertion that is missing in other MBOAT proteins1. The DltB–DltC interface is mostly hydrophobic, formed by DltB residues Met302, Val305, Ile306 and Met309, and DltC residues Met36, Val39, Val43 and Val55. In addition, Arg317—the first residue of helix H14—forms charged hydrogen bonds with DltC Glu40, whereas the phosphate group of Ser35-Ppant is in a position to form a salt bridge with DltB Lys282 in helix H12 (Fig. 2d, Extended Data Figs. 5, 7). The structures of DltB are essentially identical in both the apo state and the DltC-bound state (Extended Data Fig. 7a).

To confirm the structural and functional features of the DltB–DltC interface, we purified DltB mutants V305D and V305D/I306D as well as DltC mutants V39D and V39R, and tested their interactions with their corresponding wild-type partner using GST pull-down and Octet assays (Fig. 2b, Extended Data Fig. 6). Whereas DltB(V305D) showed substantially reduced binding to wild-type DltC, the binding was completely abolished when using DltB(V305D/I306D). Similarly, both DltC(V39D) and DltC(V39R) showed substantially reduced ability to interact with DltB. These mutagenesis analyses demonstrate that Val305 and Ile306 of DltB and Val39 of DltC are critical to the DltB–DltC interaction, and confirm our structural observation that these surface residues are located at the DltB–DltC core interface.

There is an approximately straight tunnel between the bottom of the extracellular funnel and the cytoplasmic side. This tunnel is formed by three DltB helices from the C-ridge (H13–H15) and the small horizontal helix H12 from the central core. DltB residues inside the tunnel are highly conserved among DltB proteins (Fig. 3a, Extended Data Fig. 5), and show a level of conservation in other MBOAT proteins, which suggests that this tunnel is functionally important. It should be noted that in our current structures of DltB and the DltB–DltC complex, the side chain of the conserved Trp285 from helix H12 keeps this tunnel in a closed conformation (Fig. 3a, Extended Data Fig. 7c). We speculate that the conformation we captured is that of the DltB enzyme in a ‘resting’ state.

Fig. 3: Structure of the DltB tunnel and the DltB–DltC-Ppant binding mode provide insight into the molecular mechanism of DltB.
figure 3

a, Residues forming the DltB tunnel. b, Cut-away surface illustration of the DltB–DltC-Ppant complex. DltC-Ppant pSer35 is located at the bottom of the tunnel. c, LTA d-alanylation assay. m-AMSA is a DltB inhibitor. The assays were repeated three times. H281, S285, H328 and V297/F298 in B. subtilis correspond to H289, S293, H336 and V305/I306 in S. thermophilus, respectively. d, Lysozyme susceptibility survival assay. Representative images are shown for serial dilutions of cells plated on LB agar (left) and LB agar supplemented with 30 µg ml−1 of lysozyme (right). Dilutions of cells are indicated on the y axis. The survival assay was performed three times. e, A working model for DltB-mediated LTA d-alanylation. Cross-membrane d-alanylation is probably mediated by the DltB tunnel, the opening (activation) of which may be controlled by helix H12. The role of DltD in this reaction is unclear.

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One notable feature of the DltB–DltC complex is that DltC Ser35 is located at the cytoplasmic entrance of the tunnel (Fig. 3b). Whereas the electron density for the Ppant phosphate group is well-defined in our electron density map, the density for the rest of the Ppant chain is too thin for model building; this is consistent with a ‘resting’ state conformation. Consistently, Octet analysis showed that the DltC(S35A) mutant can also interact with DltB with similar affinity (Kd ≈ 0.19 µM) to that of wild-type DltC-Ppant, which indicates that the Ppant group is not essential to the DltB–DltC interaction. The Ppant group can potentially switch between occupying the tunnel and being flexible in the cytoplasmic open space, as the Ser35 phosphate group is positioned between the tunnel entrance and the open cytoplasmic space. While the most conserved residue (His336) is located at the C terminus of the DltB H14 helix, DltC makes contacts with the C-terminal half of the H13 helix and the N terminus of the H14 helix—which suggests that the distance between DltC Ser35 and DltB His336 may be largely fixed during catalysis.

To examine the functional importance of the tunnel, we generated B. subtilis strains that lacked the dlt operon, and then complemented with a heterologous copy of the dlt locus expressed from its native promoter. The LTA d-alanylation level and the viability of dlt-deleted B. subtilis cells complemented with a heterologous copy of the dlt locus, containing either wild-type dltB or various dltB mutations, were evaluated using 14C- d-Ala radiolabelling and lysozyme-sensitivity assays, respectively. Mutation of DltB residues corresponding either to S. thermophilus DltB His336 or to the DltC-binding site completely abolished LTA d-alanylation (Fig. 3c). In addition, both mutations considerably reduced the viability of B. subtilis in the presence of lysozyme, whereas mutations of two other DltB residues did not have a substantial effect in both assays (Fig. 3c, d, Extended Data Fig. 8). Our functional assay data together with the structural features of DltB strongly suggest that the tunnel is important for the catalytic mechanism of DltB (Fig. 3e).

In some other O-acyltransferases, such as carnitine acyltransferase24, a conserved histidine catalyses the acyl-transfer reaction by aligning the carnitine substrate with the acyl-CoA thioester bond. The Ppant- d-Ala chain has a length of around 20 Å between the phosphate group and d-Ala. In our crystal structure, the distance between the Ser35-Ppant phosphate group and His336 is approximately 21 Å. Should the tunnel be open for the Ppant-d-Ala chain binding, this distance would enable His336 to align the substrate that receives the acyl group (probably a glycerol phosphate unit within LTA molecule) with d-alanylated DltC-Ppant. Thus, our structures suggest a model in which d-alanylation of LTA occurs between the LTA bound to the extracellular funnel and the d-alanyl group on DltC-Ppant-d-Ala bound to the cytoplasmic side of the tunnel (Fig. 3e).

Because DltB forms a stable complex with DltC-Ppant even without the d-alanyl group, and the DltC Ser35 is open to the cytosol, we speculate that DltC-Ppant forms a constitutive complex with DltB during catalysis and the Ppant chain can migrate between the tunnel and the cytosol, where loading of the d-alanyl group of DltC-Ppant can be catalysed by DltA. We then asked how the DltB tunnel opens for Ppant binding. The DltB tunnel is formed by the small horizontal helix H12 and the long transmembrane helices (H13–H15) forming the C-ridge of DltB. Compared to the DltB C-ridge helices, helix H12 is more likely to be the mobile structural component. The tunnel opening can be caused by movement or by a conformational change of the short helix H12, the position of which is stabilized through local hydrophobic interactions. H12 may change its position without disturbing the N- and C-ridge structures and lead to the opening of the tunnel. H12 movement may be induced by the presence of an appropriate signal, such as substrate binding with the extracellular funnel and/or binding of intracellular ligands such as DltC-Ppant- d-Ala. It should be noted here that in the Dlt system DltD is required for the d-alanylation of LTA in vivo. It remains unclear how DltD may contribute to this process. A combination of structural and enzymatic analysis is needed to reveal the role of DltD and the detailed catalytic mechanism of DltB.

We next considered the implication of the DltB structure for other MBOAT proteins. Despite a low overall sequence homology, a more conserved region within MBOAT sequences—termed MBOAT2 homology—was identified (http://pfam.xfam.org/family/MBOAT_2). The MBOAT2 homology covers the sequences that correspond to the DltB region from DltB H12 to the N terminus of H15 (Fig. 1b), which forms the majority of the central core that is thinner than the lipid bilayer. Thus, the thin central core and the extracellular (or lumen-facing) funnel are likely to be common structural features in many MBOATs. It has been demonstrated that the most conserved histidine (DltB His336), which is located within this MBOAT2 homology domain, is critical for the enzymatic activities of all tested MBOAT proteins—including PORCN, HHAT, GOAT, ACAT and DGAT17,18,19,20,21,22—which strongly suggests a common or similar catalytic mechanism for the MBOAT superfamily of proteins. The conserved extracellular/lumen structural funnel, the thin central core and the tunnel that we observed in our DltB structure are probably shared by many other MBOATs. Indeed, our crystal structure of DltB is in good agreement with the membrane topology models of HHAT and GOAT that have previously been derived from biochemical data25,26,27 (Extended Data Fig. 9). For example, in each case, the catalytic histidine was predicted to be at the end of an HHAT or a GOAT transmembrane helix facing the lumen side, consistent with our structure of DltB. That the critical horizontal helices H11–H13 in our DltB core structure were predicted to be a cytoplasmic subdomain of HHAT or GOAT is also consistent with models. In addition, a predicted ‘re-entrant helix’ observed in both HHAT and GOAT corresponds to the H7–H8 ‘half-way turn-back’ structure in the DltB central core (Fig. 1b, Extended Data Fig. 9). In contrast to the similarity in the core structure, the N- and C-terminal regions of HHAT and GOAT are much more divergent.

Among vertebrate MBOATs, PORCN and GOAT are responsible for lipid modifications of secreted Wnts and ghrelin, respectively. They all catalyse reactions across the endoplasmic reticulum membrane, with the acyl-group-accepting proteins located in the endoplasmic reticulum lumen and acyl-CoA in the cytosol6,7. Because DltB also catalyses cross-membrane reactions, we examined the sequence homology among DltB, PORCN and GOAT. It appears that there are four conserved regions: the region covering DltB helices H12–H14 (the MBOAT2 homology region), the DltB H7–H8 region, and two partial helices in the inner circle of the DltB structure (most of helix H6 and the central part of helix H10) (Fig. 4, Extended Data Fig. 9). Therefore, although sequences encoding the N- and C-ridges of DltB are generally not conserved in other MBOATs, the central core of DltB—along with its structural neighbours in the inner circle (for example, parts of helices H6 and H10)—are conserved among vertebrate MBOATs, including PORCN and GOAT. We suggest that the non-conserved nature of the ridges enable recognition of distinct substrates specific to different members within the MBOAT family. It should be noted that the mechanism of acyl-group binding is probably very different between metazoan MBOATs, most of which bind acyl-CoA as a donor, and DltB, which uses DltC-Ppant-d-Ala.

Fig. 4: Conserved regions among bacterial DltB and vertebrate PORCN and GOAT proteins.
figure 4

a, Alignment of conserved regions of DltB, PORCN and GOAT. Conserved sequences are highlighted in yellow (and in green under the sequences). The red rectangle indicates the DltB-specific insertion, which is involved in the binding of DltC-Ppant. A red star marks the most conserved histidine residue among MBOATs. St, S. thermophilus; Lc, Lactobacillus casei; Hs, Homo sapiens; Xl, Xenopus laevis; Mm, Mus musculus. b, The conserved MBOAT core. Conserved regions among MBOATs form a central core in the DltB structure (coloured in green), whereas the peripheral helices shielding the core are largely non-conserved (coloured in wheat).

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The deep, conserved DltB extracellular structural funnel, as well as the DltB tunnel, may be an excellent target for drug development. Furthermore, many other bacterial and metazoan MBOATs may also be very druggable targets, as many of them are present on the surface of the cell membrane. In addition, the deep extracellular/lumen funnel shape close to the active site is probably a conserved feature of many MBOATs, and may be an excellent drug-binding site. Indeed, even in the absence of a 3D structure and detailed enzymatic analysis using purified PORCN, multiple small-molecule inhibitors with half-maximal inhibitory concentrations in the low-nanomolar range have been found through cell-based screening, and some of them have been used in clinical trials for the treatment of cancer28,29,30. Potent HHAT and GOAT inhibitors have also been reported and examined in several studies5. On the basis of our crystal structures, we predict that many more highly potent MBOAT inhibitors will be discovered in the future.

Three-dimensional structural prediction of MBOAT proteins has been very difficult and unreliable. Our crystal structures of DltB serve as a cornerstone for understanding the structure and function of MBOAT proteins. In addition, our structures reveal an intriguing mechanism for cross-membrane catalysis, and provide a platform for the development of new clinically relevant drugs across species.

Methods

Protein preparation

The cDNA of full-length S. thermophilus DltB was subcloned into pET21b (Novagen). cDNAs of S. thermophilus AcpS, DltA and DltC were subcloned into pQLink vectors (Addgene) with AcpS and DltA bearing an N-terminal 6×His-tag, and DltC bearing a N-terminal GST-tag. Escherichia coli strain C43 (DE3) was used for protein overexpression. Overexpression of the above proteins was induced by 0.4 mM isopropyl β-d-thiogalactoside when cell density reached an optical density at 600 nm (OD600) of 1.0. After induction at 37 °C for 5 h, the cells were collected and homogenized in buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl.

For DltB purification, after disruption by sonication, cell debris was removed by centrifugation for 10 min at 20,000g. The supernatant was collected and ultracentrifuged for 1.5 h at 100,000g. The membrane fraction was collected and homogenized with buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl. n-Decyl-β-d-maltopyranoside (Anatrace) was added to the membrane suspension to a final concentration of 1.5% (w/v) and then incubated for 2 h at 4 °C. After another ultracentrifugation step at 100,000g for 30 min, the supernatant was collected and loaded onto Ni-NTA affinity resin (Ni-NTA; Qiagen). After washing with buffer containing 25 mM Tris-HCl pH 8.0, 500 mM NaCl, 25 mM imidazole and 0.2% (w/v) n-decyl-β-d-maltopyranoside, DltB was eluted with a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 400 mM imidazole and various detergents from Anatrace. After being concentrated to 10 mg ml−1, DltB was further purified by gel filtration (Superdex-200 10/30; GE Healthcare). The buffer for gel filtration contained 25 mM Tris-HCl pH 8.0, 150 mM NaCl and various detergents. The peak fractions were collected.

For the purification of DltA and DltC, after sonication the cell debris was removed by centrifugation for 1 h at 35,000g. The supernatants were loaded onto Ni-NTA affinity resin and Glutathione Sepharose 4 resin (GS4B resin, GE Healthcare), respectively. After a wash step, the N-terminal GST-tag was either removed from DltC or maintained, depending on the purpose of the experiment. After elution, DltA and DltC solutions were loaded onto HiTrap Q HP columns (5 ml, GE Healthcare), and protein samples eluted from the Q column were further purified by gel filtration. Peak fractions were collected and concentrated. Finally, DltA and DltC were stored in buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl.

DltB and DltC mutants were generated with a standard PCR-based strategy and were subcloned, overexpressed and purified in the same way as the wild-type proteins.

Protein crystallization

The hanging-drop vapour-diffusion method was performed at room temperature during crystallization. DltB and DltC proteins were purified as mentioned above, and crystals were obtained from DltB purified with n-nonyl-β-d-glucopyranoside (Anatrace). For crystallization of the DltB–DltC complex, DltB and DltC were purified separately and mixed before crystallization at a molar ratio of 1:2. Crystals belonging to crystal form I (space group P21, Extended Data Table 1) were crystallized in buffer containing 21% PEG400, 100 mM Tris-HCl pH 7.5, 100 mM NaCl and 100 mM MgCl2. Crystals belonging to crystal form II (space group P21, Extended Data Table 1) were crystallized in buffer containing 27% PEG400, 100 mM sodium citrate pH 5.6, 200 mM NH4H2PO4 and 100 mM (NH4)2SO4. Crystals belonging to crystal form III (space group P212121, Extended Data Table 1) were crystallized in buffer containing 27% PEG400, 100 mM HEPES pH 7.5, 200 mM sodium citrate tribasic dihydrate and 3% 1,5-diaminopentane dihydrochloride. For crystals in the different crystal forms above, thin or thick rod-shaped crystals typically grew for 1 to 2 weeks before reaching full crystal size. Gold derivatives were obtained by soaking the crystals in crystal form I for 2 h in mother liquor containing 2 mg ml−1 KAu(CN)2.

Data collection and structure determination

The crystals were directly flash-frozen in liquid nitrogen. Screening and data collection were performed at the Advanced Light Source, beamlines 5.0.1, 8.2.1 and 8.2.2. All diffraction data were processed by HKL200031. The single-wavelength anomalous dispersion (SAD) dataset was collected near the gold L-III absorption edge at a wavelength of 1.02 Å (Extended Data Table 1). The gold derivative sites and the initial phases were determined by PHENIX32. Twenty gold derivative sites were found in one asymmetric unit, and the experimental electron density map clearly showed the presence of four DltB molecules in one asymmetric unit. The B. subtilis DltC crystal structure (PDB ID: 4BPH) was used as the searching model for our DltC molecules33. The complex model was improved using iterative cycles of manual rebuilding with the program Coot34 and refinement with a native dataset of 3.30 Å using Refmac5 of the CCP4 7.0 program suite35. The structures for crystal forms II and III were solved by molecular replacement using the model from crystal form I. All structure model figures in the paper were generated using PyMOL36. The protein conservation surface was generated using the ConSurf server37, based on the alignment of DltB sequences generated using T-Coffee38.

Binding assay

Pull-down assays were performed as described below. Twenty micrograms of wild-type DltB (or DltB mutants), 10 µg of wild-type GST–DltC (or GST–DltC mutants) and 10 µl GS4B resin were mixed in 100 µl of pull-down buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl and 0.15% (w/v) n-decyl-β-d-maltopyranoside. The mixed samples were incubated at 4 °C on a rotisserie for 1 h, followed by washing the resin with pull-down buffer three times. During each wash, 100 µl of pull-down buffer was added to each sample and the solution was incubated at room temperature for 2 min before centrifugation and removal of supernatant. After washing, the resin samples were analysed by SDS–PAGE with Coomassie blue staining.

Binding assays were also performed at room temperature using the Octet system (FortéBio). Free GST, and GST-tagged wild-type DltC or DltC mutants were mobilized on anti-GST biosensors (FortéBio). After quenching with free GST to block free antibody sites on the biosensors, the biosensors were dipped into DltB solutions for binding measurements. The concentration gradient of DltB used in the Octet binding assay is: 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, 10 µM.

Construction of B. subtilis strain for functional assays

The cat gene was amplified by PCR from pGEMcat, and 500 bp upstream and downstream of dlt operon fragments were amplified from the B. subtilis genome. These three pieces were assembled using isothermal assembly and transformed directly into the B. subtilis HM1 strain, resulting in dlt-operon-deleted B. subtilis (∆dlt). The deletion was confirmed by PCR amplification and Sanger sequencing.

The natural dlt locus was amplified and cloned into pMMB752. Mutations of the dltB gene in pMMB752 carrying the dlt operon and Flag-tagged constructs were generated on the basis of a standard PCR method, followed by isothermal assembly to ligate the ends together. The pMMB752 constructs were transformed into B. subtilis with the dlt operon deleted from its native locus to generate strains for use in assays. Cells used here and in the following functional experiments were cultured in the presence of appropriate antibiotics to avoid possible contamination.

Detection of LTA d-alanylation

This assay was established on the basis of a previously reported method14. Wild-type B. subtilis HM1 strain, and dlt-operon-deleted B. subtilis HM1 strain complemented with either empty pMMB752 vector or vectors containing natural dlt-operon-bearing mutations on the dltB gene (untagged or Flag-tagged), were inoculated from fresh colonies on plate into liquid LB medium supplemented with 0.5 µg ml−1 erythromycin. Overnight cultures were diluted into 3 ml of LB at an OD600 of 0.1 and grown to an OD600 of 0.6. Cells were pelleted and resuspended into 1.5 ml of assay medium containing 0.25× LB, 50 mM Bis-Tris pH 6.0, and 200 µg ml−1 d-cycloserine. To test the inhibition of m-AMSA on LTA d-alanylation for wild-type B. subtilis, a final concentration of 150 µM of m-AMSA (Abcam) was supplemented into the assay medium. After incubation in the assay medium for 30 min, 14C-d-alanine (Moravek Biochemicals) was added to a final concentration of 25 μM for an additional incubation of 30 min or 120 min. Cells were pelleted and resuspended with SDS-loading buffer, followed by a freeze–thaw cycle. Samples were vortexed and boiled for 5 min before loading onto 4–20% gradient Tris/glycine gel (Bio-Rad). Gels were dried and exposed to a phosphor storage screen for 3 days before imaging with Typhoon FLA 9000 gel imaging scanner (GE Healthcare).

To compare the expression level of C-terminal Flag-tagged DltB in corresponding B. subtilis strains, each strain was cultured in 1 l LB to OD600 of 0.6. Cells were collected and disrupted by French press, and the cell membrane was isolated by ultracentrifugation after removing cell debris by low-speed centrifugation. The membrane of each strain was resuspended with buffer containing 25 mM Tris-HCl 8.0, 150 mM NaCl into 500 µl, followed by freezing at −80 °C. One microlitre of each membrane sample was run onto SDS–PAGE and the expression of Flag-tagged DltB was detected by western blotting.

Survival assays

Dlt knockout strains of the Gram-positive bacterium B. subtilis are sensitive to the cell-wall-degrading enzyme lysozyme39. B. subtilis strains were struck on LB plates (supplemented with the appropriate antibiotic when needed) from freezer stocks and incubated overnight at 37 °C. The resulting growth on plates was used to inoculate 2-ml LB broth cultures in glass tubes. The cultures were grown at 37 °C with shaking (260 r.p.m.) to an OD600 of 1.0–2.0. All of the cultures were adjusted to an OD600 of 0.3 and then serially diluted in LB broth with tenfold dilutions. For each strain, 5 µl of each dilution was plated onto LB plates and LB plates supplemented with 30 µg ml−1 of lysozyme (Fisher) and incubated at 30 °C overnight. After incubation, colonies were enumerated and plates were imaged with a Bio-Rad Gel Doc XR+ Molecular Imager.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.