Abstract
Cyclic-di-AMP (c-di-AMP) is an essential second messenger in Bacillus subtilis and many other Gram-positive bacteria. Work over the past decade has revealed that this cyclic nucleotide controls cation and osmolyte transporters, leading to the hypothesis that c-di-AMP regulates cytoplasmic turgor pressure. Although the targets of c-di-AMP are well established, the signals that control the levels of this second messenger and the factors that transduce these signals are unknown. Here we report that c-di-AMP levels are modulated by the cyclase regulator CdaR in response to cell wall defects. We further demonstrate that changing the levels of c-di-AMP alters turgor pressure. Our data support a model in which CdaR senses defects in the cell wall and activates c-di-AMP synthesis in response. The increase in c-di-AMP reduces turgor, preventing lysis and enabling fortification of the peptidoglycan meshwork. Thus, a central function of c-di-AMP is to control cellular turgor in response to envelope defects.
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Data availability
No large-scale datasets were generated over the course of this study. All relevant data are available in the supplementary material. Raw data for all graphs and uncropped immunoblots have been uploaded as source data. Primers, synthetic DNA constructs and strains used can be found in supplementary tables. The RefSeq database downloaded from NCBI (https://ftp.ncbi.nih.gov/refseq/release/) as of June 2019 was used for the described bioinformatics analysis. The phylogenetic tree was generated from 5,767 unique bacterial taxa assembled from the assembled reference genomes in the prokaryotic RefSeq database as of June 2019. Source data are provided with this paper.
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Acknowledgements
We thank A. Gründling and all members of the Bernhardt–Rudner supergroup for helpful advice, discussions and encouragement, and the MicRoN core for advice on fluorescence microscopy. Electron Microscopy Imaging and services were performed in the HMS Electron Microscopy Facility. Portions of this research were conducted on the O2 High Performance Computing Cluster, which is supported by the Research Computing Group at Harvard Medical School. Support for this work was provided by National Institute of Health Grants R35GM145299, U19 AI158028 (D.Z.R.) and R35GM143057 (E.R.R.). A.P.B. was funded in part by NSF (DGE1745303) and NIH (F31AI181098).
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A.P.B. and D.Z.R conceived the study. A.P.B. and P.B. performed the experiments and analyses. D.Z.R and E.R.R. supervised the study. A.P.B. and D.Z.R wrote the paper, with edits from P.B. and E.R.R.
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Extended data
Extended Data Fig. 1 Cells with reduced levels of c-di-AMP are more sensitive to envelope-targeting antibiotics, while cells with increased levels of c-di-AMP are more resistant.
Representative growth curves of the indicated strains grown in LB with 5, 50 µM, or 500 µM IPTG treated with sub-inhibitory concentrations of the indicated antibiotics. (A) Red lines show cells that express low levels of CdaA with 5 µM IPTG and grey lines show cells that express wild-type levels of CdaA with 50 µM IPTG. 50 µM IPTG is closest to physiological expression levels (Extended Data Fig. 3e). (B) Blue lines show cells that express high levels of CdaA with 500 µM IPTG in the absence of the phosphodiesterases GdpP and PgpH. All experiments in this figure were performed in biological triplicate and representative growth curves are shown.
Extended Data Fig. 2 Analysis of growth and c-di-AMP levels in mutants and after antibiotic exposure.
(A) Bar graph showing β-galactosidase activity from the riboswitch reporter of the indicated B. subtilis deletion mutants. Cells lacking cdaAR have lower c-di-AMP and cells lacking gdpP have higher c-di-AMP. All measurements were performed in biological triplicate except the ∆cdaS condition which was performed in duplicate. (B) Bar graph showing β-galactosidase activity from the kimA reporter in cells lacking disA, cdaS, and cdaAR, and harbouring an IPTG-regulated allele of cdaAR in the presence of 50 µM IPTG. The strain was treated with sub-inhibitory concentrations (0.5X MIC) of fosfomycin (FOS), bacitracin (BAC), penicillin G (PENG), oxacillin (OX), vancomycin (VAN), moenomycin (MOE), D-cycloserine (DCYC). Cells only increase c-di-AMP levels in the presence of moenomycin that inhibits the glycosyltransferase activity of class A PBPs. (C) Bar graph showing β-galactosidase activity from the kimA riboswitch reporter in cells expressing CdaAR or DisA as the only c-di-AMP cyclase in the presence of absence of PBP1. The center of error represents the average, and the error bars represent the standard deviation across three biological replicates. (D) 10-fold serial dilutions of the indicated strains spotted on LB agar supplemented with the indicated IPTG concentrations. CdaAR is critical in the absence of PBP1.
Extended Data Fig. 3 Analysis of CdaA and CdaR variants.
(A) Immunoblot of B. subtilis cells expressing cdaR-His at its native locus (first lane) or cdaR-His and variants at an ectopic location under IPTG-control with 15 µM IPTG. All strains lack disA and cdaS. (B) Immunoblot of cells expressing CdaA-His under IPTG-inducible control with 500 µM IPTG and CdaR-HA or CdaR∆ID-HA under xylose-inducible control with 30 mM xylose. cdaA and cdaR were inserted at separate genomic location. The strains express (+) or lack (∆) PBP1. (C) Cells expressing CdaR-His at its native locus (lanes 1 & 2) compared to CdaA-CdaR-His with and without its IDR (∆ID) under IPTG-inducible control at an ectopic locus with 50 µM IPTG. (D) Immunoblots from a co-immunoprecipitation assay using anti-His resin and detergent-solubilized membrane fractions. The load (L) and eluate (E) were probed for CdaA-His, CdaR-HA, CdaR∆ID-HA and the membrane protein EzrA. The B. subtilis strains expressed CdaA with or without a His-tag under IPTG-inducible control with 500 µM IPTG and CdaR-HA with or without its IDR under xylose-inducible control with 30 mM xylose. A non-specific cross-reactive band is indicated (*). The CdaA:CdaR complex does not require CdaR’s IDR. The co-IP was performed in biological duplicate and a representative experiment is shown. (E) Immunoblot of B. subtilis cells expressing CdaR-His at its native locus (lanes 1&2) or CdaA-CdaR-His with and without its IDR under IPTG-inducible control expressed with the indicated IPTG concentrations. SigA was used to control for loading in panels (A), (B), (C), and (E). 50 µM IPTG is closest to physiological expression of CdaA and CdaR. All Immunoblots are representative of three biological replicates.
Extended Data Fig. 4 An acute increase in c-di-AMP causes membrane invaginations.
(A) Growth curve of cells containing Phy-cdaS(L44F) induced with 500 µM (pink) or without (black) IPTG. (B) Representative fluorescence images of cells grown in the presence of 500 µM IPTG for the indicated times. Cells were harvested at the indicated times, but images were acquired ~5 minutes later due to cell concentration, mounting, and imaging. Cells contain constitutively expressed cytoplasmic mCherry and membranes are labeled using the membrane protein SpoIVFB-GFP (FB-GFP). Sites of cytoplasmic retraction and membrane invagination are highlighted (yellow carets). (C) The same experiment as described in (B) with membranes stained with the membrane dye TMA-DPH. Scale bars indicate 5 µm.
Extended Data Fig. 5 Analysis of c-di-AMP levels in cells expressing cdaS(L44F) or gdpP(cyto).
(A, B) Bar graphs showing β-galactosidase activity from the kimA riboswitch reporter in B. subtilis strains expressing cdaS(L44F) from (A) the Pspank (PIPTG) or (B) the Phyperspank (Phy) promoters. Expression was induced with 500 µM IPTG for 90 minutes. (C) Bar graph showing c-di-AMP levels as measured by ELISA assay in the same conditions as shown in (A, B). Values were normalized to the culture OD600. All ELISA measurements were performed in biological triplicate except the Phy condition which was performed in duplicate. (D) Bar graph showing β-galactosidase activity from the kimA reporter of cells expressing Phy-gdpP(cyto). Samples were taken at the indicated timepoints after addition (+) of 500 µM IPTG. (E) Bar graph showing c-di-AMP levels normalized to OD600 as measured by ELISA after 45 minutes of induction with Phy-gdpP(cyto). In all panels, the center of error represents the average, and the error bars represent the standard deviation across three biological replicates.
Extended Data Fig. 6 An acute increase in c-di-AMP causes membrane invaginations.
Transmission electron micrographs of exponentially growing cells harboring Phy-cdaS(L44F) before and at indicated timepoints after the addition of 500 µM IPTG. Cells were immediately fixed after harvest. Scale bars indicate 100 nm. Sample preparation and imaging were performed in biological duplicate and representative images are shown.
Extended Data Fig. 7 An acute increase in c-di-AMP levels causes membrane invaginations independent of phospholipid biosynthesis.
Representative images of B. subtilis cells harboring Phy-cdaS(L44F). Cells were induced with 500 µM IPTG for 10 min and then treated with cerulenin (12.5 µg/mL) to block phospholipid synthesis for 5 min prior to harvest. Images were acquired ~ 5 minutes later due to cell concentration, mounting, and imaging. The delay in cerulenin addition (10 min after IPTG-induction) was chosen based on control experiments showing that the drug rapidly inhibits transcription from an IPTG-regulated promoter (Supplementary Fig. 6). Importantly, no membrane invaginations are detectable at 10 min after IPTG addition (Extended Data Fig. 6). Thus, cerulenin treatment at 10 minutes enabled cdaS(L44F) expression and inhibition of phospholipid biosynthesis prior to the generation of membrane invaginations. Membrane accumulation did not increase over the next 75 minutes in cerulenin treated cells, suggesting that the severity of the membrane invaginations is partially due to uncoupled phospholipid synthesis from growth. Cells constitutively expressed cytoplasmic mCherry and were stained with TMA-DPH to label membranes. Sites of membrane invagination in the presence or absence of cerulenin are highlighted (yellow carets). Scale bar indicates 5 µm.
Extended Data Fig. 8 Membrane invaginations caused by an acute increase in c-di-AMP depend on the external osmolarity.
(A) Bar graph showing the osmolarity of LB and LB lacking salt (no salt) as measured using an osmometer. The center of error represents the average and error bars indicate the standard deviation among three replicates. (B) Representative fluorescence micrographs of cells harboring Phy-cdaS(L44F) before (-IPTG) or at indicated time points after the addition of 500 µM IPTG ( + IPTG). 10 or 15 min after induction, cells were pelleted, resuspended in LB or LB (no salt), spotted onto LB or LB (no salt) 2% agarose pads and imaged 5 min after harvest. Cells contain constitutively expressed cytoplasmic mCherry and membranes were labeled using a constitutively expressed membrane protein SpoIVFB-GFP (FB-GFP) and the membrane dye TMA-DPH. Carets highlight sites of cytoplasmic retraction and membrane invagination. Low external osmolarity (LB no salt) largely suppresses the membrane invagination caused by high c-di-AMP. Scale bars indicate 5 µm.
Extended Data Fig. 9 An acute decrease in c-di-AMP causes bulging and lysis.
(A) Growth curves of B. subtilis cells harboring Phy-gdpP(cyto) that lacks its transmembrane and PAS domains induced with 500 µM IPTG (red line) or without inducer (black line). (B) Representative fluorescence and phase-contrast images of Phy-gdpP(cyto) cells at the indicated time points in the presence or absence of 500 µM IPTG. Cells were harvested at the indicated timepoints, and images were acquired ~5 minutes after harvest due to cell concentrating, mounting, and imaging. Cells constitutively express cytoplasmic mCherry. Sites of cell bulging (yellow carets) and lysis (white carets) are indicated. Scale bar indicates 5 µm.
Extended Data Fig. 10 Cyclic-di-AMP controls cellular turgor.
(A) Diagram of a cell inflated by turgor pressure P = RT(Cin-Cout), where Cin is the cytosolic osmolarity and Cout is the extracellular osmolarity. Adapted from Bardetti et al36. (B,C,D) Osmotic force extension curves and lysis strains of cells lacking disA, cdaS, cdaAR and harboring PIPTG-cdaAR grown with 5 µM IPTG (B), wild-type (WT) (C), and cells with PIPTG-cdaS(L44F) grown with 500 µM IPTG (D). Cells grown in LB in a microfluidic chamber were exposed to a hyperosmotic shock of LB containing the indicated molarity of sorbitol (x-axis), and the mean percent change in cell length (εl) in response to the shock [(lengthfinal-lengthinitial)/lengthfinal] was measured. The solid horizontal line indicates the average change in cell length (Lysis strain) upon lysis with detergent. Lysis strain was determined by growing cells in LB in a microfluidic chamber and measuring the percent change in cell length [(lengthfinal-lengthinitial/lengthfinal] before and after exposure to LB containing 5% of the anionic detergent N-lauroylsarcosine. Dotted lines represent the linear regression used to find pressure, which was calculated as the shock magnitude that causes contraction of length to the rest length upon cell lysis. The colored dotted lines indicate the pressure (P) in units of molarity shown in Fig. 5k. For the lysis experiments, n = 136,82,116 for LOW, WT, and HIGH, respectively. For the hyperosmotic shock experiments, the n for increasing sorbitol concentration was n = 104,104,104,68(LOW), n = 116,116,116,376(WT), and n = 50,50,50,59,74(HIGH). Error bars indicate average longitudinal strain and standard error among replicates. (E) Spot dilutions on LB, LB (no salt), or LB ( + 500 mM sorbitol) agar all containing 15 µM IPTG. Strains lack disA, cdaS, and cdaAR and harbour the indicated IPTG-regulated allele of cdaAR. Images are shown after one and two days of incubation. High osmolarity suppresses the growth defects associated with cdaAR-∆ID expression in the absence of PBP1. (F) Bar graph showing the osmolarities of the indicated media as measured by an osmometer. The center of error represents the average and error bars represent the standard deviation among three replicates. (G) Bar graph showing β-galactosidase activity from the kimA riboswitch reporter of strains assayed in (A) grown to mid-log in LB ( + 500 mM sorbitol). The strains lack pbp1, disA, cdaS, and cdaAR and express the indicated cdaAR allele with 50 µM IPTG. High external osmolarity does not impact the intracellular c-di-AMP levels of the strains. The center of error represents the average and error bars indicate the standard deviation between two biological replicates. (H) Growth curves of the indicated strains in LB and LB ( + 500 mM sorbitol). High osmolarity suppresses the growth defect associated with cdaA mutants. Spot titers and growth curves are representative of three biological replicates.
Supplementary information
Supplementary Information
Supplementary Text and Figs. 1–8.
Supplementary Table 1
Strains used in the study.
Supplementary Table 2
Plasmids used in the study.
Supplementary Table 3
Oligonucleotides used in the study.
Supplementary Table 4
GBlock sequences used in the study.
Supplementary Video 1
Time course of lysozyme treatment from Fig. 5i.
Supplementary Data 1
Numerical source data separated by figure for the supplementary figures.
Source data
Source Data Figs. 1, 2, 4 and 5, and Extended Data Figs. 1, 2, 4, 5 and 8–10
All numerical source data in tabs labelled by figure panel.
Source Data Figs. 1–3 and Extended Data Fig. 3
Unprocessed western blots labelled by figure panel.
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Brogan, A.P., Bardetti, P., Rojas, E.R. et al. Cyclic-di-AMP modulates cellular turgor in response to defects in bacterial cell wall synthesis. Nat Microbiol 10, 1698–1710 (2025). https://doi.org/10.1038/s41564-025-02027-2
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DOI: https://doi.org/10.1038/s41564-025-02027-2
