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Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan

Nature Microbiology volume 5, pages 291–303 (2020)Cite this article

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Abstract

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton1. In Staphylococcus aureus, the peptidoglycan assembly enzymes relocate during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells2,3,4. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood5,6. Here, we used a transposon screen to identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes stem peptides from uncrosslinked peptidoglycan and a partner protein that controls its activity. Amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so that they can separate7. However, this amidase controls cell growth. In its absence, peptidoglycan synthesis becomes spatially dysregulated, which causes cells to grow so large that cell division is defective. We show that the cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given subcellular location. Removal of stem peptides from peptidoglycan at the cell periphery promotes peptidoglycan synthase relocation to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.

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Fig. 1: LytH is an amidase involved in cell growth and division.
Fig. 2: The LytH–ActH complex cleaves stem peptides from uncrosslinked peptidoglycan.
Fig. 3: Slowing peptidoglycan synthesis compensates for loss of LytH.
Fig. 4: LytH spatially regulates peptidoglycan synthesis.

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Data availability

Transposon sequencing data (accession numbers SAMN08025141 and SAMN08025168) can be found in the NCBI BioSample database. Whole-genome sequencing data (accession number PRJNA579395) can be found in the NCBI BioProject database. All other data are available in the manuscript, Extended Data, Supplementary Information or Source Data.

Code availability

Code is available at https://github.com/SuzanneWalkerLab/imageanalysis.

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Acknowledgements

The authors thank M. Welsh and A. Taguchi for help with LC–MS analysis and protein expression. They thank T. Pang from the Bernhardt and Rudner laboratories for strain TD215. Fluorescence images were partially acquired at the MicRoN core at Harvard Medical School. The authors acknowledge support from the National Science Foundation (grant no. DGE1144152 to T.D.), the National Institutes of Health (grant nos. P01AI083214, R01GM076710, R01AI139011, R01AI099144), and the European Research Council (grant no. ERC-2017-CoG-771709 to M.G.P).

Author information

Authors and Affiliations

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

    Truc Do, Kaitlin Schaefer, Ace George Santiago, Kathryn A. Coe & Suzanne Walker

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Kaitlin Schaefer & Daniel Kahne

  3. Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal

    Pedro B. Fernandes & Mariana G. Pinho

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Contributions

S.W. and T.D. conceived the project, designed experiments and analysed the data with advice from M.G.P on acquisition and analysis of fluorescence images. T.D. performed all experiments except the radiolabelled gel assays, which were performed by K.S., and some of the microscopy experiments, which were performed by P.B.F. A.G.S. and T.D. developed scripts for image analysis. K.C. analysed the transposon sequencing data. S.W., D.K. and T.D. wrote the manuscript with input from all authors.

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Correspondence to Suzanne Walker.

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Extended data

Extended Data Fig. 1 Deletion of lytH sensitizes cells to oxacillin and causes growth defects.

a, HG003 strains were spotted on TSA containing 0.1 μg/mL oxacillin and 0.4 μM of the inducer, anhydrotetracycline (atc), when indicated. Strains expressing lytH or gfp from an atc- inducible promoter in a ∆lytH background are denoted by plytH or pgfp, respectively. b, Transmission electron micrographs: Additional examples of defects in placement of nascent septa observed for the ∆lytH mutant. Scale bars, 500 nm. c, Growth was assessed by measuring optical density (left graph) and colony-forming units (right graph). Data represent mean ± SD. Error bars that are smaller than the dots are not shown. Data are representative of two (a-b) and three (c) independent experiments.

Extended Data Fig. 2 Muropeptide analysis identifies a tetrasaccharide- monopeptide.

a, Schematic of sacculi isolation and mutanolysin/NaBH4 treatment to generate muropeptides for LC-MS analysis. b, Total ion chromatograms comparing the muropeptide species isolated from WT vs. ∆lytH sacculi. Muropeptide species eluted between 12-18 min using this analytical method. The XCMS online platform was used to identify species that were enriched or depleted between the two strains. c, The tetrasaccharide-monopeptide species was enriched in sacculi isolated from cells expressing lytHWT. A chemical structure consistent with this species, based on an [M + 2] ion with experimental m/z = 866.3881, is shown. d, The [M + 2] ion was targeted for fragmentation to confirm the species was a tetrasaccharide-monopeptide. e, The theoretical and experimental isotope distributions for the tetrasaccharide- monopeptide species are shown.

Extended Data Fig. 3 LytH pulled down ActH, a previously uncharacterized polytopic membrane protein.

a, A homology model of LytH highlights the predicted catalytic residues identified by sequence alignment of S. aureus LytH with E. coli AmiC. b, TMHMM topology prediction suggests that LytH is anchored in the cytoplasmic membrane. c, Schematic representation of the BDL-labeling assay to assess LytH hydrolytic activity in vitro. Short glycan strands were synthesized from Lipid II using S. aureus SgtBY181D. E. faecalis PBPX was used to exchange the terminal amino acid of stem peptides for biotin-D-Lys (BDL, yellow spheres), enabling visualization of glycan strands by western blotting with streptavidin. d, Glycan strands synthesized in vitro were treated with LytH. No difference in the distribution or intensity of polymers was observed between the three lanes. Blot is representative of two independent experiments. e, SDS-PAGE analysis of the co-immunoprecipitation experiment to identify proteins that bound to full-length LytH. Purple asterisks mark the protein bands corresponding to the baits. A red box is drawn around the ActH protein band. ActH is encoded by the gene saouhsc_01649. The fractions are: (FT) flowthrough, (W1-W2) washes, and (E1-E2) elutions. The schematics for LytH constructs are shown with the predicted transmembrane (TM), SH3, and amidase catalytic domains annotated. The predicted domain structure of ActH is also shown. f, A cladogram based on 16S rRNA alignment of bacterial species that encode ActH homologs with a similar domain structure was generated. The tree shows 196 bacterial species. For ease of visualization, only some species are labeled. Firmicutes are highlighted in purple and Deltaproteobacteria are highlighted in red.

Source data

Extended Data Fig. 4 The LytH-ActH complex has peptidoglycan hydrolase activity.

a, HG003 strains were spotted on TSA ± 0.125 μg/mL oxacillin. b, The relative ion count (extracted ion count/total ion count) for the tetrasaccharide-monopeptide was calculated. Data represent the mean ± SD. P-values were determined using unpaired, two-tailed t-tests (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). From left to right: P = 0.0048, 0.0005, 0.0387, 0.0010, 0.0580, and 0.0056. c, Full-length LytH and ActH were co-expressed in E. coli and purified from detergent-solubilized membranes as a stable complex. SDS- PAGE analysis of purification fractions: (FT) flowthrough. d, Western blot analysis of LytH reactions with glycan strand substrate, showing a distinct difference for the WT complex. Glycan strands were made in vitro from native S. aureus Lipid II; the strands were labeled with BDL for visualization. e, Western blot analysis of LytH reactions with glycan strand substrate, showing the catalytic serine of ActH is dispensable for activating LytH. The bold bands appearing in all lanes in the blot correspond to purified proteins that were added to the reactions and non-specifically labeled. Data are representative of three (b-c) and two (a, d-e) independent experiments.

Source data

Extended Data Fig. 5 The LytH-ActH complex removes stem peptides from uncrosslinked glycan strands.

a, Chemical structure highlighting the differences between synthetic and S. aureus native Lipid II. b, PAGE autoradiographs of reactions with uncrosslinked glycan strand substrates. The glycopolymers for substrates (1) and (3) were made from synthetic Lipid II, while the glycopolymers for substrate 2 were generated from native Lipid II. For substrate (1), the radiolabel was found in the [14C]-GlcNAc residues of the glycan backbone. For substrates (2) and (3), the radiolabel was provided by a short wall teichoic acid (WTA) disaccharide branch ([14C]-LIIAWTA) attached to the hydroxyl group at the C6 position. c, Purified S. aureus sacculi were treated with lysostaphin and LytHWT-ActH. Hydrolysis of sacculi was monitored over time as a decrease in OD600. Experiments were performed in triplicate for each concentration of protein; each line represents the mean, plotted as percent of initial absorbance. d, Schematic for a PAGE assay to assess if LytH-ActH cleaves crosslinked peptidoglycan. Uncrosslinked glycan strands (substrate 2) were made from native Lipid II and radiolabeled with a short [14C]-LIIAWTA disaccharide branch (substrate 1) at the C6 position. The radiolabeled glycan strands were crosslinked by S. aureus PBP4 to generate crosslinked peptidoglycan (substrate 3). e, Peptidoglycan substrates were treated with LytHWT-ActH or lysostaphin. Reaction products were resolved by PAGE. Lysostaphin cleaves crosslinks, generating smaller species that migrate faster in the gel. Molecular weight ladders were not run on these PAGE gels. Data are representative of three independent experiments (b-c, e).

Source data

Extended Data Fig. 6 Mass spectrometry confirms LytH-ActH amidase activity.

a, Glycan strands were made from native Lipid II using SgtBWT protein and treated with the LytH complex prior to LC-MS analysis. In the presence of LytHWT-ActH, the tetrasaccharide-monopeptide was observed, but only the monomer (starting material) was observed in the LytHD195A-ActH reaction. We could not detect the released stem peptide in these reactions with native glycan strands likely due to the poor ionization efficiency of the released pentaglycine-containing stem peptide. b, The chemical structure and isotope distributions for the disaccharide peptide species (blue peak) shown in Fig. 2f. c, The chemical structure and isotope distributions for the synthetic stem peptide product (red peak) shown in Fig. 2f.

Extended Data Fig. 7 PBP2 suppressor mutants showed reduced peptidoglycan synthesis activity.

a, Spot dilutions showing that excess PBP2 is toxic in the absence of LytH. Wild-type pbp2 (pbp2WT) or a pbp2* suppressor allele (pbp2N220→KDLN) was expressed from an inducible promoter in strain HG003 ΔlytH. In these strains, the native pbp2WT allele was still present. b, Fluorescence images of Nile red-stained PBP2 overexpression strains. The controls in which the inducer was withheld to prevent PBP2 overexpression are shown. Quantitation of cellular phenotypes is summarized in the graph below: cells with a nascent or complete septum at midcell (Class A), cells showing only membrane fluorescence (Class B), and cells showing fluorescent punctate foci or multiple septa (Class C). Scale bars, 1 µm. c, A PBP2 suppressor variant was purified. Bocillin- labeling of purified proteins showed that PBP2F158L was properly folded. The Coomassie gel was a control for amounts of protein loaded. The experiment was performed once. d, Western blot comparing PBP2WT and PBP2F158L reactions with Lipid II substrate. PBP2 polymerizes Lipid II into glycan strands and crosslinks the glycan strands; PBP2 also incorporates BDL to enable visualization of crosslinked peptidoglycan. The PBP2 reactions were incubated for 5 or 15 min. When indicated, lysostaphin was added to cleave peptidoglycan crosslinks so that highly-crosslinked material could enter into the gel. Data are representative of two independent experiments (a-b, d).

Source data

Extended Data Fig. 8 Reducing PBP2 activity suppresses lytH deletion defects.

a, Fluorescence images of Nile red-stained HG003 strains. The suppressor allele pbp2F158L (pbp2*) mitigates ∆lytH division defects. Quantitation of cellular phenotypes is shown in the graph to the right: cells with a nascent or complete septum at midcell (Class A), cells showing only membrane fluorescence (Class B), and cells showing multiple septa (Class C). Scale bars, 1 µm. b, Deletion of lytH leads to larger cell size. The distribution of cell volume was calculated for cells at different phases of growth: cells that have not initiated septal synthesis (phase 1), cells with a nascent septum (phase 2), and cells with a complete septum prior to splitting into two daughter cells (phase 3). Each dot represents a single cell (from left to right): n = 566, 516, 615, 259, 160, 201, 215, 145, and 241 cells. The median of each spread is indicated by a black line. P-values were determined by two-sided Mann-Whitney U tests (***P < 0.001). From left to right: P = 4.58x10-50, 8.10x10-86, 2.21x10-26, 1.94x10-30, 2.44x10-21, and 2.70x10-25. c, Transmission electron micrograph of the suppressor strain HG003 ∆lytH pbp2F158L is shown. Scale bar, 500 nm. The experiment was performed once at 30 °C and 37 °C. d, The number of cells with septal defects was counted. Class A cells showed normal placement of a nascent or complete septum at midcell, while Class B cells showed misplaced septa. Data are representative of two independent experiments (a-b).

Source data

Extended Data Fig. 9 Loss of LytH reduces peptidoglycan synthesis at the septum relative to the periphery.

a, Fluorescence images of fluorescein-D-lysine (FDL)-labeled cells. All FDL labeling was performed in strains lacking PBP4 to reduce background. Cells were sorted into different classes according to the pattern of FDL signal: at the septum (Class A), around the membrane (Class B), or at misplaced septa and/or peripheral puncta (Class C). Scale bars, 1 µm. b, The ratio of fluorescence intensity at the septum vs. periphery was calculated for cells with a single complete septum. Two regions of interests, outlined in red, were defined to retrieve pixel values at the periphery and the septum. c, Deletion of lytH leads to reduced septal to peripheral FDL signal independently of cell size. Each dot represents a single cell: n = 159 (wt) and 167 (∆lytH) cells. d, FDL fluorescence ratio is not correlated with cell size (n = 105 cells). Deletion of ezrA causes division defects and larger cell size. e, Fluorescence images of an FDL-labeled, mixed population of WT and ∆lytH cells. To distinguish WT from ∆lytH cells, one strain was stained with DAPI prior to mixing the two strains and labeling with FDL. Reciprocal labeling of DAPI was performed. Scale bars, 1 µm. f, FDL signal for mixed populations containing DAPI-stained WT cells. Fluorescence intensity measurements correspond to the median intensity at the periphery or septum. Different colored dots represent cells analyzed from different fields of view (sets 1-3): n = 52 (wt) and 17 (∆lytH) cells. P-values were determined by two-sided Mann-Whitney U tests (***P < 0.001; ns, not significant). From left to right: P = 0.9389 and 7.02x10-5. g, Fluorescence images of cells labelled with fluorescent vancomycin (Van-FL) to detect newly synthesized peptidoglycan. Scale bars, 1 μm. Data are representative of two independent experiments (a-g).

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Extended Data Fig. 10 PBP2 was delocalized in the absence of LytH.

a, Fluorescence images of NCTC8325-4 strains expressing sGFP-PBP2. To compare the spatial distribution of PBP2 signal in WT vs. ∆lytH cells, fluorescence ratios were calculated using cells with a single complete septum. In all plots, each dot represents a single cell, the median of each spread is indicated by a black line, and p-values were determined by two-sided Mann-Whitney U tests. In this plot: n = 81 (wt) and 77 (∆lytH) cells; P = 8.30x10-8 (***P < 0.001). Scale bars, 1 µm. b, Fluorescence images of strain NCTC8325-4 ∆ezrA expressing sGFP-PBP2, showing that delocalization of PBP2 is independent of cell size. Fluorescence ratios of sGFP-PBP2 signal was plotted against cell volume (n = 91 cells). Scale bars, 2 µm. c, Fluorescence images of HG003 strains co-expressing sGFP-PBP2 and FtsZ-mCherry. PBP2 and FtsZ signals showed reduced colocalization in ∆lytH cells. Only cells showing FtsZ signal at the septum were considered for Pearson correlation coefficient analysis to calculate the degree of colocalization: n = 133 (wt) and 166 (∆lytH) cells; P = 0.006 (**P < 0.01). Scale bars, 1 µm. Different fields of view were captured for one biological sample (a-b). Data are representative of two (c) independent experiments.

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Do, T., Schaefer, K., Santiago, A.G. et al. Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan. Nat Microbiol 5, 291–303 (2020). https://doi.org/10.1038/s41564-019-0632-1

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