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Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus

Abstract

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

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Fig. 1: Caulobacter growth in glucose is inversely controlled by (p)ppGpp and c-di-GMP.
Fig. 2: Competitive binding of c-di-GMP and ppGpp to the second messenger binding protein SmbA.
Fig. 3: SmbA is a structural homologue of AKRs with overlapping binding sites for c-di-GMP and ppGpp.
Fig. 4: C-di-GMP binding induces loop ordering and helix α9 reorientation.
Fig. 5: SmbA-mediated growth control.
Fig. 6: C-di-GMP and (p)ppGpp control growth on glucose by balancing the cellular redox state.
Fig. 7: C-di-GMP and (p)ppGpp inversely control SmbA activity to mediate Caulobacter surface attachment.

Data availability

Data can be accessed via the Source Data files and Supplementary Tables. Mass spectrometry data are deposited on the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifiers PXD019846 and https://doi.org/10.6019/PXD019846. The Protein Data Bank accession numbers for the coordinates of the structures reported here are 6GS8 and 6GTM. Source data are provided with this paper.

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Acknowledgements

We thank T. Sharpe (Biophysics Facility, Biozentrum, University of Basel), A. Schmidt and T. Bock (Proteomics Core Facility, Biozentrum, University of Basel), J. Bögli and S. Stefanova (FACS Core Facility, Biozentrum, University of Basel) for their technical guidance, F. Hamburger for cloning and strain construction, B. Lehtinen for help with the metabolite sampling, K. Sprecher and I. Hug for strain construction, and C. v. Arx for plasmids. We thank R. Hallez and S. Crosson for providing published plasmids. This work was supported by a European Research Council (ERC) Advanced Research Grant (grant no. 3222809 to U.J.), the Swiss National Science Foundation (grant nos. 310030B_147090 (to U.J.), 31003A_166652 (to T.S.) and 31003A_173094 (to J.A.V.)).

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Conceptualization: V.S., B.N.D., T.S. and U.J. Methodology: V.S., B.N.D, J.N., R.B., S.H., J.A.V. and J.H. Formal analysis: V.S., B.N.D., R.B., J.H., T.S. and U.J. Investigation: V.S., B.N.D., R.B., J.H., J.N., T.S. and U.J. Resources: U.J. and T.S. Writing of the original draft: V.S., B.N.D., T.S and U.J., with contributions from all other authors. Funding acquisition: T.S. and U.J.

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Correspondence to Tilman Schirmer or Urs Jenal.

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Extended data

Extended Data Fig. 1 Caulobacter growth on glucose is inversely regulated by (p)ppGpp and c-di-GMP.

(a) Growth of Caulobacter wild type and Δrel mutant inoculated from PYE stationary phase cultures into fresh M2G. For Δrel, adaptation periods and logarithmic growth are separated by a dashed red line. Growth curves were parameterized (adaptation phase t0, exponential growth rate k) by fitting a corresponding mathematical function to the data (see Methods). (b) and (c) Growth of Caulobacter wild type and mutant strains inoculated from logarithmically growing (b) and stationary phase cultures (c) into fresh M2G. Shown are mean of n = 3 biological replicates ± SD. (d) and (e) Ten-fold serial dilutions of Caulobacter wild type and mutant strains from exponentially growing (d) or stationary phase cultures (e) on solid MG2 agar plates. (f) Phase contrast microscopy images (left panel) and quantification of cell length (right panel) for Caulobacter wild type and Δrel mutant during adaptation and exponential growth phase. (g) Re-passaging experiments confirm the stability of the Δrel growth phenotype. The process of growth and re-plating is shown schematically. (h) and (i) Growth of Caulobacter rel + (h) and Δrel mutant (i) at gradually increasing c-di-GMP concentrations. (j) Growth of Caulobacter wild type and mutant strains inoculated from PYE stationary phase cultures into fresh M2X. (k) Uptake of 3H-glucose by Caulobacter wild type and mutant strains as indicated. The proton motive force inhibitor CCCP was used to block sugar uptake. (l) Transcription of the zwf operon as measure by lacZ reporter fusions in strains indicated grown in M2G. (m) Activity of the zwf promoter was determined as in (l) for the strains indicated. Pink and green dotted lines represent reporter activity in Caulobacter wild type grown in M2G and M2X, respectively. In panels (g-m), data represent the mean of n = 3 biological replicates ± SD.

Source data

Extended Data Fig. 2 Oxidative stress detection in Caulobacter.

(a) RSG fluorescence of Caulobacter wild type challenged with different levels of hydrogen peroxide for 10 min during exponential growth in M2G was measured by FACS. (b) Top: Schematic representation of Grx1-roGFP redox sensitive probe activation. Bottom: Exponentially growing Caulobacter wild type cultures expressing were treated with 10 mM hydrogen peroxide or 10 mM DTT and analysed by FACS. (c) The indicated Caulobacter strains expressing Grx1-roGFP were grown to the early adaptation phase in M2G and 405/488 nm excitation ratios were calculated as shown in (b). (d) Schematic of DCF-DA fluorescent probe activation. (e) ROS detection with the DCF-DA fluorescent probe in strains indicated during logarithmic growth and stationary phase in PYE or during adaptation in M2G. Shown are mean of n = 3 biological replicates ± SD.

Source data

Extended Data Fig. 3 Binding parameters of SmbA protein in complex with nucleotides.

(a-j) Isotherms representing binding of recombinant SmbA wild type or mutant proteins with c-di-GMP (a-d), ppGpp (e-h), pppGpp (i) and GMP (j) as measured by ITC. Please see Supplementary Table 2 for detailed experimental conditions and measured parameters.

Extended Data Fig. 4 Effects of second messenger binding to SmbA monitored by NMR spectroscopy.

(a) 2D [15N,1H]-TROSY spectrum of 160 μM [U-15N]-labelled SmbA in the apo state (magenta). (b) Spectral overlay of 160 μM apo SmbA (magenta) and 160 μM SmbA + 800 μM ppGpp (orange). (c) Spectral overlay of 160 μM apo SmbA (purple) and 160 μM SmbA + 300 μM c-di-GMP (light blue). (d) Spectral overlay of 160 μM SmbA + 300 μM c-di-GMP (blue) and 160 μM SmbA + 800 μM ppGpp (orange). All experiments were recorded in 30 mM HEPES pH 7.0, 250 mM NaCl, 5 mM MgCl2, 1 mM DTT measured at 20 °C on a 700 MHz spectrometer. The spectral region surrounded by the black box is enlarged in Fig. 2d.

Extended Data Fig. 5 Structural details of second messenger binding to SmbA.

(a) Cartoon representation of the (c-di-GMP)2 complex with full structural details of the ligands and interacting residues. H-bonds (length < 3.5 Å) and cation - π interactions are indicated by red and magenta lines, respectively. Note that R133*, which forms lateral H-bonds with O6 and N7 of g2 and cation - π interaction with g1, is donated by an adjacent protomer in the crystal packing (see Supplementary Fig. 7a). (b) Structure of the ppGpp complex. Note that the C-terminal carboxylate of a symmetry related protomer is forming a salt-bridge with R251 (see Supplementary Fig. 7b). (c) 2Fo-Fc omit maps contoured at 1.2 σ of (c-di-GMP)2 (left) and ppGpp (right) as bound to SmbA. (d) Fo-Fc omit maps contoured at 3.0 σ of (c-di-GMP)2 (left) and ppGpp (right).

Extended Data Fig. 6 (p)ppGpp and c-di-GMP inversely control SmbA activity to promote growth on glucose.

(a) Growth of Caulobacter wild type and different smbA mutants in M2G media. (b) Growth of Caulobacter wild type and mutants indicated in the rel + background diluted from stationary phase cultures (PYE) into fresh M2G containing 50 mM sodium chloride. The rcdG0::dgcZ strain harbours an IPTG-inducible (100 µM) copy of dgcZ on the chromosome. (c) Growth of Caulobacter wild type and Δrel mutant in M2X media supplemented with 50 mM sodium chloride. (d) Growth of Caulobacter Δrel mutants containing different smbA mutant alleles (as indicated) in M2G media supplemented with 30 mM sodium chloride. In panels (a-d), data represent the mean of n = 3 biological replicates ± SD. (e) SmbA specifically promotes growth on M2G. Caulobacter mutants smbA Q114A and smbA R143A harbouring plasmid pSA280 (Plac-dgcZ; 150 μM IPTG) were grown on minimal glucose (M2G), minimal xylose (M2X) and complex media (PYE) as indicated. Ratios of doubling times are indicated as a measure for the growth promoting role of SmbA under different conditions. Shown are the mean of n = 3 biological replicates ± SD. (f) Viability of Caulobacter Δrel mutants harbouring different smbA alleles was assayed by diluting cultures from stationary phase (PYE) onto solid MG2 agar plates. (g) Immunoblots of Caulobacter cell extracts (wild type and Δrel mutant) were stained using polyclonal anti-SmbA antisera. Cultures were grown as indicated in Fig. 1b and reached stationary at 15 hours. Similar results were obtained in three independent experiments.

Source data

Extended Data Fig. 7 (p)ppGpp and c-di-GMP control growth on glucose by balancing cellular redox state.

(a) Relative levels of radicals as measured by DCF fluorescence intensity in the strains indicated. Fluorescence was scored in exponentially growing cells in M2X (left bar) or in M2X supplemented with 10 mM ascorbic acid (right bar). Dashed lines indicate the levels of DCF fluorescence measured in Caulobacter wild type treated with 10 mM ascorbic acid (green) or untreated (black). (b–e) Growth of Caulobacter wild type and indicated mutant strains. Cells were diluted from cultures growing exponentially in PYE to fresh M2G or M2X with or without 10 mM ascorbic acid as indicated. In panels (a-e), data represent the mean of n = 3 biological replicates ± SD. (f) Flow cytometry analysis of Caulobacter wild type and mutant cells grown on M2X. Cells were sampled after growth in M2X for 15 h as indicated in (d) and (e) and samples were incubated with RSG for 20 min before FACS analysis. (g) Caulobacter cell cycle progression is affected by (p)ppGpp and SmbA. The optical density of synchronized populations of wild type and mutant strains was scored during one cell cycle as indicated. (h) Immunoblot analysis of synchronized populations indicated in (g) using polyclonal anti-CtrA antisera. CtrA protein levels are used as a marker for cell cycle progression (indicated for Caulobacter wild type above panels in g and h). Similar results were obtained in three independent experiments.

Source data

Extended Data Fig. 8 SmbA regulates Caulobacter surface attachment.

(a) Fraction of Caulobacter wild type and smbA mutants with visible holdfast. (b) Representative microscopy images (overlay of phase contrast and fluorescent GFP channels) of cultures with stained holdfast (indicated by red arrows) used for calculations described above. For each strain, similar results were obtained from three independent cultures. (c) Transcription of hfiA in Caulobacter wild type and mutant strains as indicated. A lacZ transcriptional reporter was used to determine hfiA promoter strength. β-galactosidase activities were determined in strains containing different smbA alleles on the chromosome. A staR deletion mutant was used as a control. (d) Attachment of hfiA deletion strains harbouring different smbA alleles as measured by crystal violet stain. In panels (a, c-d), data represent the mean of n = 3 biological replicates ± SD.

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Supplementary information

Supplementary Figs. 1–5, Supplementary Tables 2, 4–7 and Supplementary Methods.

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Supplementary Table 1

List of all peptides identified from the CCMS for cdG effector screen (S1) and mass spectrometry-based proteome analysis (S3).

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Statistical source data for Supplementary Fig. 4.

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Original autoradiographs from three independent experiments.

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Representative original unmodified gels.

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Representative original unmodified gels.

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Shyp, V., Dubey, B.N., Böhm, R. et al. Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus. Nat Microbiol 6, 59–72 (2021). https://doi.org/10.1038/s41564-020-00809-4

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