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Two broadly conserved families of polyprenyl-phosphate transporters

Nature volume 613, pages 729–734 (2023)Cite this article

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Abstract

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1,2,3,4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5,6,7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

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Fig. 1: UptA and PopT complete the lipid II cycle.
Fig. 2: DedA and DUF368 family members provide resistance to UndP-targeting antibiotics.
Fig. 3: Gene neighbourhood analyses for DedA and DUF368 family members.
Fig. 4: UptA and PopT are broadly conserved UndP transporters.

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Data availability

Next-generation sequencing datasets generated in this study have been uploaded to the NCBI Sequence Read Archive within project PRJNA898639 (SAMN31619234 and SAMN31619233). Reads were mapped to the B. subtilis genome (NCBI NC_00964.3). Next-generation sequencing datasets that were reanalysed from ref. 16 are available on the NCBI Sequence Read Archive (SRX3390726), and the analysis was conducted as described in the paper. Uniprot accession codes for gene neighbourhood analysis are provided in Supplementary Table 3. Details of the strains, plasmids, oligonucleotides and synthetic DNA constructs used are provided in Supplementary Tables 4–7. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Bernhardt–Rudner super-group for helpful advice, discussions and encouragement, T. Bernhardt, S. Walker and A. Kruse for insights and advice, S. Farmer and B. Hancock for MX2401, the MicRoN core for advice on microscopy, and M. Waldor for coordinating submission. Support for this work comes from the National Institute of Health grants GM086466, GM127399, GM145299 and U19 AI158028 (D.Z.R.).

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

    Ian J. Roney & David Z. Rudner

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  1. Ian J. Roney
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  2. David Z. Rudner
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Contributions

I.J.R. and D.Z.R. conceived of the study and I.J.R. carried out all experiments and analysis. D.Z.R. supervised the study. I.J.R. and D.Z.R. wrote the paper.

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Correspondence to David Z. Rudner.

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Extended data figures and tables

Extended Data Fig. 1 Polyprenyl-phosphate lipids are universal transporters of surface glycopolymers.

Schematic illustrating the glycopolymers and sugar modifications that are assembled on undecaprenyl-phosphate (UndP) in bacteria and on dolichol-phosphate (DolP) in archaea and eukaryotes. Although an oligosaccharide carried on DolP is shown, some eukaryotes use the pyrophosphate carrier (DolPP).

Extended Data Fig. 2 YngC has features of membrane transporters and is expressed under σM control.

Structural models of YngC (a) and SAOUHSC_00846 (d) as predicted by AlphaFold2. (Left) Membrane re-entrant helices are highlighted in red and blue. Conserved arginines in the red re-entrant helix of YngC are indicated. (Right) Surface charge distribution in the predicted structures. (b) Minimum inhibitory concentration (MIC) assays of the indicated B. subtilis strains with point mutations in the conserved arginines in yngC. (c) Immunoblot analysis of YngC-His levels using anti-His antibodies of the strains in (b). SigA is the sample processing control. (e) Schematic of the yngABC operon highlighting the promoters that regulate yngC expression. Two are recognized by sigma factor A (σA) and one is recognized by the ECF sigma factor M (σM). (f) Representative fluorescence images of cells harboring a transcriptional fusion of the yngC σM promoter to yfp. YFP fluorescence increases in cells exposed to vancomycin for 30 min, a condition that activates σM. The reporter is not induced by vancomycin in a ∆sigM mutant. Scale bar, 1 µm. (g) Quantification of YFP fluorescence from 100 cells of the strains and conditions in (f). Bar represents the median. (h) Streaks of the indicated strains on LB agar supplemented with 1.25 µg/mL MX2401. P∆sigM contains a deletion of the σM promoter of yngC. (i) The Tn-seq screen for MX2401 resistance mutants identified insertions in the genes (yhdL and yhdK) encoding the anti-σM factors, consistent with increased σM-dependent transcription of yngC providing MX2401 resistance. Transposon insertion profile at the indicated B. subtilis genomic region is shown. Each vertical line indicates an insertion site; its height reflects the number of reads at this position (maximum height ≥5,000). The transposon insertion site with the maximum number of reads in this region had 2,600 reads. For comparison, the insertion sites adjacent to yngC had >20,000 reads.

Source data

Extended Data Fig. 3 ∆yngC sensitizes B. subtilis to reduced levels of UndP synthesis.

(a) Spot-dilutions of the indicated B. subtilis strains with IPTG-regulated alleles on LB agar supplemented with the indicated concentrations of IPTG. Strains with reduced levels of IspH or UppS are sensitive to the absence of yngC. Relevant comparisons are boxed in red. Strains with reduced levels of RodA, MurG or MraY grow similarly in the presence or absence of yngC. (b) Schematic of the UndP synthesis pathway illustrating the two sources of UndP for lipid II biogenesis: de novo synthesis and recycling. Enzymes shown in red were expressed at reduced levels in the assays in (a). (c) Minimum inhibitory concentration (MIC) of MX2401 in the indicated B. subtilis strains, each lacks one of the six DedA paralogs. (d) The B. subtilis ∆yngC mutant was mutagenized with a transposon carrying a strong outward facing Ppen promoter (insert). The library was plated on LB agar supplemented with 0.3 µg/mL MX2401 to select for mutants that provide resistance. Transposon insertion profiles at the indicated B. subtilis genomic regions are shown. Each vertical line indicates an insertion site; its height reflects the number of sequencing reads at this position (maximum height ≥5000). The average number of reads was >40,000. The majority of insertions mapped upstream of the ykoX gene in an orientation that would increase its transcription. Transposon insertions were not enriched upstream of bcrC or uppP that encode UndPP phosphatases, suggesting that these proteins do not have UndP transport activity, as was proposed previously3,38. Unlike the Tn-seq screen in a wild-type (yngC+) background, in the ∆yngC mutant, transposon insertions were not enriched in the genes (yhdL and yhdK) encoding the anti-σM factors, consistent with the model that their inactivation provides increased MX2401 resistance by increasing σM-dependent transcription of yngC.

Extended Data Fig. 4 A transposon-sequencing screen identified insertions upstream of 02816 and 00846 that confer resistance to amphomycin in S. aureus.

(a) Reanalysis of Tn-seq data from Santiago et al. 201816. A library of S. aureus mutagenized with a transposon carrying a strong outward facing promoter (Ptuf) was grown in sub-inhibitory concentrations of amphomycin (9.6 µg/mL). Transposon insertion profiles at the indicated S. aureus genomic regions are shown. Each vertical line indicates an insertion site; its height reflects the number of reads at this position (maximum height ≥2,000). Most transposon insertions mapped upstream of SAOUHSC_02816 and SAOUHSC_00846 in orientations that are predicted to increase transcription of these genes. By contrast, insertions were not enriched upstream of the UndPP phosphatase uppP, suggesting that this membrane phosphatase does not have UndP transport activity, as previously proposed3,38. (b) Representative micrographs of wild-type and the ∆2 (∆uptA ∆popT) double mutant. Shown are overlays of phase-contrast and fluorescence images of propidium iodide-stained cells. Quantification of the PI-positive cells from several fields of view (>1,000 cells per strain) yielded a PI-positive rate of 0.1% for wild-type and 10% for the ∆uptA ∆popT mutant. (c) Wild-type and ∆2 cells were stained with fluorescent membrane dyes FM-464 (red) and TMA-DPH (blue), respectively, and then mixed and imaged on the same agarose pad. Scale bar, 1 µm. (d) Spot-dilutions of the indicated S. aureus strains harboring an empty vector (EV) or a vector with an IPTG-regulated promoter fused to uppS or murA. The ∆2 (∆00846 ∆02816) and ∆3 (∆00846 ∆02816 ∆00901) strains have an aTc-regulated allele of 00846 (846). In the presence of 500 µM IPTG the ∆2 strain overexpressing UppS grows similarly to wild-type and the ∆3 strain is able to form tiny colonies at all dilutions. By contrast, the growth of the two mutants overexpressing MurA phenocopies the mutants with the empty vector. Overexpression of MurA enables growth on LB agar plates supplemented with 3 µg/mL fosfomycin.

Extended Data Fig. 5 Gene neighborhood analyses reveal that dedA paralogs can be found adjacent to or fused with pap2 lipid phosphatases or adjacent to uppP undecaprenyl-pyrophosphate phosphatases.

Representative genomic neighborhood analyses showing synteny (a) and gene-fusions (b) of DedA family members with PAP2 lipid phosphatases. (c) Schematic of the lipid II cycle highlighting the role of PAP2 family members like BcrC and DedA family members like YngC (UptA) in dephosphorylating UndPP and flipping UndP across the membrane, respectively. (d) Representative genomic neighborhood analysis showing synteny of dedA transporters with the undecaprenyl-pyrophosphate phosphatase uppP. Most examples of synteny between dedA and uppP genes are from Streptomycetes and Paenibacilli genomes. (e) Schematic of the lipid II cycle highlighting the role of UppP family members and DedA family members like YngC (UptA) in dephosphorylating UndPP and flipping UndP across the membrane, respectively. MIC assays of the indicated B. subtilis strains lacking uptA and ykoX (∆2) expressing one of the eight Bacillus simplex (f) or Paenibacillus cellulosyliticus (g) DedA paralogs. BS18575 is adjacent to a pap2 gene in B. simplex. PC16690 is adjacent to a uppP gene in P. cellulosyliticus. Strains were grown in LB with 500 µM IPTG in the presence of the indicated concentration of MX2401. Uniprot IDs for the proteins included in the genome neighborhood diagrams can be found in Supplementary Table 3.

Extended Data Fig. 6 Gene neighborhood analysis indicates that duf368 genes are often present in archaeal gene clusters involved in S-layer protein glycosylation.

(a) Representative genomic neighborhood from Haloferax volcanii with all the characterized genes involved in S-layer protein glycosylation and popT highlighted. (b) Schematic of the S-layer protein glycosylation pathway encoded in the H. volcanii gene cluster as described in ref. 26. PopT is hypothesized to catalyze the recycling of DolP to complete the cycle. AglR is shown flipping one of the two UndP-linked sugars but could transport both. (c) Gene neighborhood analysis showing synteny of duf368 (popT) transporters with aglB and other genes involved in N-linked protein glycosylation (outlined in black). Uniprot IDs for the proteins included in this diagram can be found in Supplementary Table 3. (d) Cartoon depiction of the regulation of the yngABC and ykcBC operons by the transcription factor YclJ54. (e) Schematic of the putative cell surface glycosylation pathway encoded by YclJ regulon members27. YngA is a member of the GtrA flippase family that transports UndP-linked monosaccharides across the cytoplasmic membrane. YngB is a member of the UDP glucose pyrophosphorylase family and has been shown to charge sugars with UDP groups27. YkcB is a member of the glycosyltransferase-39 family that transfers monosaccharides from the UndP carrier to surface polymers and is thought to glycosylate lipoteichoic acid (LTA). YkcC is a member of the glycosyltransferase-2 family that adds UDP charged monosaccharides onto UndP on the cytoplasmic leaflet of the membrane. The DedA paralog YngC (UptA) is proposed to complete the cycle.

Extended Data Fig. 7 MX2401-FL specifically labels outward-facing UndP.

(a) Validation of MX2401 conjugated to CF488 (MX2401-FL). Representative fluorescence and phase-contrast images of the indicated strains labeled with MX2401-FL and propidium iodide (PI). Cells with reduced de novo synthesis of UndP (UppS LOW) harbor an IPTG-regulated allele of uppS (Pspank-uppS) and were propagated in the presence of 4 µM IPTG. Cells with increased de novo synthesis of UndP (UppS HIGH) harbor a stronger IPTG-regulated allele of uppS (Physpank-uppS) and were propagated with 500 µM IPTG. UppS LOW cells have low MX2401-FL staining and bulge due to impaired for cell wall synthesis. UppS HIGH cells have high MX2401-FL staining and are shorter. Cells treated with vancomycin for 5 min prior to MX2401-FL staining trap UndP in lipid II and have low MX2401-FL signal. All MX2401-FL images were normalized identically with minimum and maximum intensities of 125 and 600 to detect weak MX2401-FL staining in the UppS LOW and vancomycin-treated strains. (b) Wild-type and UppS LOW strains were stained with MX2401-FL or a mixture of MX2401-FL and duramycin, which generates pores in the membrane allowing MX2401-FL access to the cytoplasmic-facing UndP. Cells with membrane permeability defects as assayed by PI have higher MX2401-FL signal. Cells with intact membranes (yellow carets) have lower MX2401-FL staining. All MX2401-FL images were normalized identically with minimum and maximum intensities of 125 and 1500 to prevent saturating the MX2401-FL signal. (c) Representative microscopy images of the indicated strains. Strains expressing different fluorescent proteins (B. subtilis) or labeled with different fluorescent D-amino acids (S. aureus) were mixed and then stained with MX2401-FL. (Left) overlays of phase contrast and fluorescent images in the red and blue channels to distinguish the two strains. (Right) MX2401-FL staining. Yellow carets highlight wild-type cells or cells over-expressing UptA(Bs) or PopT(Sa). White carets highlight cells lacking the UndP transporters. Scale bar, 1 µm.

Extended Data Fig. 8 UptA and PopT expression reduces MX2401-FL surface labeling.

(a) Representative fluorescence and phase-contrast images of the indicated B. subtilis strains labeled with MX2401-FL and propidium iodide. The strains with IPTG-regulated popT and uptA alleles were grown in the presence of 500 µM IPTG. (b) Quantification of MX2401-FL labeling from the strains imaged in (a). Fluorescence intensity measurements of the sidewalls of 100 cells of each genotype were determined and plotted. Bar represents the median. (c) Representative images of the indicated B. subtilis strains. Two strains expressing different fluorescent proteins were mixed and then stained with MX2401-FL. The left panels show overlays of phase contrast and fluorescent images in the red and blue channels to distinguish the two strains. The right panels show MX2401-FL staining. Yellow carets highlight cells over-expressing an UptA homolog from S. aureus (Sa), Pseudomonas aeruginosa (Pa), and E. coli (Ec) in a strain lacking uptA and ykoX (∆2). White carets highlight cells lacking uptA and ykoX. (d) Yellow carets highlight cells over-expressing a PopT homolog from Vibrio cholerae (Vc), Streptococcus pneumoniae (Sp), or Bacillus cereus (Bc) in a strain lacking uptA and ykoX (∆2). White carets highlight cells lacking uptA and ykoX. Scale bar, 1 µm.

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Extended Data Fig. 9 MX2401 conjugated to three different fluorescent dyes stains cells similarly.

Representative images of the indicated B. subtilis or S. aureus strains. Two strains expressing different fluorescent proteins (B. subtilis) or labeled with different fluorescent D-amino acids (S. aureus) were mixed and then stained with MX2401 labeled with (a) Alexa Fluor 488 (MX2401-AF488), (b) CF405 (MX2401-CF405), and (c) CF594 (MX2401-CF594). Alexa Fluor 488 prevents membrane permeation of its conjugates30,31. CF405 and CF594 are also reported to be membrane-impermeable by the manufacturer (Biotium). The left panels show overlays of phase-contrast and fluorescence images in the red and blue, red and green, or green and blue channels to distinguish the two strains. The right panels show MX2401-FL staining. Yellow carets highlight wild-type cells or cells over-expressing UptA(Bs) or PopT(Sa). All three fluorescently labeled MX2401 probes yield similar results. Scale bar, 1 µm. (d) MIC assays of the indicated strains each expressing one of the eight E. coli DedA paralogs in B. subtilis lacking uptA and ykoX (∆2). Strains were grown in LB with 10 µM IPTG. At 500 µM, cells expressing YqjA and YabI had 4-fold higher MICs (≥8-fold lower than cells expressing DedA). (e) Over-expression or deletion of the undecaprenyl-pyrophosphate phosphatase genes uppP or bcrC in B. subtilis do not alter the MIC of MX2401. MIC assays of the indicated B. subtilis strains tested with MX2401 and separately with bacitracin that targets UndPP. These experiments were performed in a strain background deleted for the bacitracin efflux pump (∆bceAB).

Extended Data Fig. 10 S. aureus and B. subtilis cells lacking UndP transporters remain polarized.

(a) Representative fluorescence and phase-contrast images of S. aureus wild-type and the ∆popT ∆uptA (∆2) mutant. The two strains were stained with the potentiometric dye 3,3′-Dipropylthiadicarbocyanine Iodide (DiSC3(5)). Treatment with the ionophore Gramicidin that depolarizes membranes was used as a positive control. The same cultures were separately stained with propidium iodide (PI) to assess the percentage of cells with membrane permeability defects. (b) Representative images of wild-type and the ∆uptA ∆ykoX (∆2) B. subtilis mutant stained with DiSC3(5). Gramicidin was used as a positive control for loss of membrane potential. Scale bar, 1 µm.

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Roney, I.J., Rudner, D.Z. Two broadly conserved families of polyprenyl-phosphate transporters. Nature 613, 729–734 (2023). https://doi.org/10.1038/s41586-022-05587-z

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