Abstract
Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR–hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces. HecE inhibits the phosphodiesterase BifA and stimulates the diguanylate cyclase WspR to increase c-di-GMP second messenger levels and promote surface colonization in a subpopulation of cells; low-level HecE-expressing cells disperse. The fraction of HecE+ cells is tuned by different stress factors and determines the balance between biofilm formation and long-range cell dispersal of surface-grown communities. We also demonstrate that the HecE pathway represents a druggable target to effectively counter P. aeruginosa surface colonization. Exposing such binary states opens up new ways to control mucosal infections by a major human pathogen.
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Data availability
The datasets generated and/or analysed during this study are available from the corresponding author on reasonable request. The raw sequencing files of the chromatin immunoprecipitation with sequencing experiment can be accessed at the NCBI under the accession number PRJNA900431. Unique biological materials are available from the corresponding author on reasonable request. The structural coordinates of the BifA R-state dimer are deposited in the PDB library under the accession number 8ARV.
Code availability
The code generated for the analysis of flow cytometry data can be accessed at https://github.com/Jenal-Lab.
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Acknowledgements
We thank F. Hamburger (Biozentrum, University of Basel) for her help with cloning. We thank E. Maffei and A. Harms (Biozentrum, University of Basel) for their support with phage isolation and characterization. This work was supported by a Biozentrum PhD Fellowship to C.M., the Swiss National Science Foundation (grant no. 310030_189253 to U.J.), the NCCR AntiResist funded by Swiss National Science Foundation (grant no. 51NF40_180541 to K.D. and U.J.), the European Research Council (grant no. 716734 to K.D.) and grants from Sygeforsikring Danmark, Danish Innovation Fund and Novoseed to M.G. and T.T.-N.
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Conceptualization: C.M., T.J. and U.J. Methodology: C.M., R.D.T., D.S., A.K., R.Z., T.J., B.-J.L., J.G.M., J.B.A., M.D., T.T.-N., K.Q., K.D. and U.J. Formal analysis: C.M., R.D.T., S.B. and D.S. Investigation: C.M., R.D.T., D.S., R.Z., T.J., J.G.M., F.W. and J.B.A. Writing (original draft): C.M. and U.J. Funding acquisition: M.G., T.T.-N., K.D., K.Q., S.H. and U.J. Supervision: S.H., K.D. and U.J.
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Extended data
Extended Data Fig. 1 The hecRE module regulates SCV and biofilm formation by altering c-di-GMP levels.
a, Colony morphology of the hecR::Tn mutant. b, Colony morphology of WT and hec deletion strains. Experiments were performed in triplicate; one representative image is shown. c, Growth of P. aeruginosa WT and hec deletion strains with mean values and the s.d. of three biological replicates (with six technical replicates each). d, Growth of WT and hec deletion strains containing plasmids for expression of hec genes from an IPTG-inducible promoter (EV, control plasmid). Growth in LB (dashed lines) or LB supplemented with 100 µM IPTG (solid lines) was recorded, and mean values and the s.d. of three biological replicates (with three technical replicates each) are shown. e, Colony morphology of P. aeruginosa WT and mutants lacking Pel or Psl exopolysaccharides expressing hecE from an IPTG-inducible promoter. Experiments were done in triplicate; one representative image is shown.
Extended Data Fig. 2 HecE modulates c-di-GMP levels by inhibiting the phosphodiesterase BifA and activating the diguanylate cyclase WspR.
a, Schematic of the BifA domain architecture and BifA fragment identified by Y2H to specifically interact with HecE (TM, transmembrane domain; SID, smallest interacting domain). b, Three-dimensional structure of a BifA dimer embedded in the cytoplasmic membrane (grey) as predicted by AlphaFold. c, CoIP-MS analysis of HecE. Immunoprecipitation experiments were performed using the PAO1 WT strain or a strain expressing C-terminally FLAG-tagged HecE and anti-M2-conjugated magnetic beads. Proteins retained on the beads were analysed using mass spectrometry. Data obtained from three biological replicates are shown as volcano plots. Log2-transformed intensity ratios of the detected peptides between HecE–Flag and the WT (ctrl) were calculated and plotted versus values derived from significance analysis (modified t-statistic, empirical Bayes method59). d, Colony morphology of P. aeruginosa wild-type and mutant strains containing an empty plasmid (EV) or a plasmid expressing hecE from an IPTG-inducible promoter. Experiments were done in triplicate; one representative image is shown. e, Growth (top) and fluorescence (c-di-GMP levels, bottom) of P. aeruginosa wild-type (WT), hecR::Tn and ΔbifA strains carrying a plasmid control (EV) or a plasmid expressing the c-di-GMP sensor cdGreen. Mean values and the s.d. of two biological replicates (with six technical replicates each) are shown.
Extended Data Fig. 3 Expression of hecR and hecE is autoregulated, bimodal and tuned by stress.
a, Expression of hecR boosts HecR and HecE levels. Immunoblot analysis of HecR and HecE in strains expressing chromosomal Flag-tagged copies of hecR or hecE. Strains contained a plasmid expressing hecR, hecE or both genes from an arabinose-inducible promoter (EV, control plasmid). Extracts of cells grown in the presence (+) or absence (−) of the inducer arabinose were analysed by immunobloting with an anti-Flag (n = 1). b, HecR binds to the hecRE promoter region. EMSA assays were carried out with labelled DNA containing the hecRE promoter region and with increasing concentrations of purified HecR protein, as indicated (n = 2). c, HecR exclusively binds to the hecRE locus on the P. aeruginosa chromosome. Chromatin pull-down experiments were performed with a strain expressing a HA-tagged copy of hecR in the absence of the chromosomal wild-type hecR copy and with HA-specific antibodies. Sequencing reads are plotted on the y axis for the entire P. aeruginosa chromosome (top) or the hecRE locus (bottom). Graphs were generated using Geneious version 2019.0 created by Biomatters. d, The fraction of cells expressing hecE changes during P. aeruginosa growth. The optical density (solid lines) and fluorescence (hecE expression; dashed lines) were recorded during the growth of hecRE-2mR reporter strains containing a plasmid expressing hecR, hecE or both genes from an IPTG-inducible promoter (EV, control plasmid). Mean values and the s.d. are shown for three biological replicates with three technical replicates each. e, The fraction of cells expressing hecRE is independent of the culture medium. P. aeruginosa wild-type carrying the hecRE-2mR chromosomal reporter was cultured in the indicated medium for 20 h. Quantification of flow cytometry data is shown for three biological replicates. Triplicates were fitted independently. Mean and range of the calculated values are shown. Normality of the means was checked usinf a Shapiro–Wilk test (P < 0.05). A one-way ANOVA test was used to analyse differences in population means (with P < 0.05), followed by Tukey’s HSD post-hoc test, for which adjusted P values are shown. f, The fraction of cells expressing hecRE varies during P. aeruginosa growth. hecRE-2mR reporter strains carrying a plasmid expressing hecR from an IPTG-inducible promoter were diluted back into fresh LB and cultured for 12 h (EV, control plasmid). Fluorescence intensities of the reporter, as measured by flow cytometry, are shown as violin plots (left y axis) and growth is indicated in stippled lines (right y axis). Cells with signal values higher than the indicated threshold (grey line) were counted as cells expressing hecRE-2mR. g, Expression of hecRE responds to nutrient limitations. The P. aeruginosa hecRE-2mR reporter strain was cultured in MOPS minimal medium with different amounts of succinate as the sole carbon source. Growth and the fraction of cells expressing hecRE are indicated as in Fig. 3f. h,i, Flow cytometry data of the hecRE-2mR reporter in ΔrsmA (h) and ΔrpoS (i) mutants, cultured in MOPS with the indicated amounts of succinate at 37 °C. Cells with higher fluorescence intensity than the PAO1 wild-type strain were quantified (right axis). The optical density of the culture before flow cytometry is indicated in stippled lines (left axis). j, Ectopic expression of hecR induces hecE transcription only in the stationary phase. Flow cytometry analysis of strains with the hecRE-2mR reporter in the wild type (WT) and indicated mutants. Reporter strains containing a plasmid expressing hecR were cultured in LB supplemented with 100 µM IPTG either to exponential phase or for 20 h (stationary). Flow cytometry experiments are shown for three biological replicates with the individual histograms stacked in the graph. k, The fraction of cells expressing hecRE is increased at elevated temperature. The P. aeruginosa hecRE-2mR reporter strain harbouring a plasmid expressing hecR from an IPTG-inducible promoter was culktured in MOPS + 20 mM succinate supplemented with 100 µM IPTG at increasing temperatures, as indicated (EV, control plasmid). Flow cytometry experiments are shown for three biological replicates with the individual histograms stacked in the graph. l, Uncropped scan of the immunoblot shown in a. m, Gating strategy used for the flow cytometry experiments. Shown is one of three replicates of the wild type carrying the hecRE-2mR reporter.
Extended Data Fig. 4 Heterogeneous expression of hecE controls surface motility, biofilm formation and dispersal.
a,d, Increase of biofilm volume of P. aeruginosa wild-type and mutant strains. Biofilms were segmented into cubes with a side length of 2.34 µm using BiofilmQ70 and biofilm volumes were calculated as the sum of all individual cubes at any given time. The red line indicates the start of the dispersal event. Analysis for one experiment is shown in each panel. b,e, Object parameters including the distance to biofilm boundary (resolution, 20 voxels), fluorescent intensities and local cell density were calculated using BiofilmQ. c,f, Fluorescent intensities at the start of the dispersal event (red line) were plotted and colour-coded according to their distance to the biofilm boundary.
Extended Data Fig. 5 HecE and H6-335-P1 antagonistically control BifA activity by influencing the T-to-R-state equilibrium.
a, Standard deviation of the mean of the attachment data shown in Fig. 5a. b, Microtiter plate-based attachment of WT and mutant strains carrying a plasmid expressing the E. coli diguanylate cyclase dgcZ from an IPTG-inducible promoter and various bifA alleles from a cumate-inducible promoter (EV, control plasmid). BifA expression was not induced, dgcZ expression was induced with 100 µM IPTG. H6-335-P1 was added at the indicated concentrations. The attachment was quantified after 9.5 h. Mean values of two biological replicates are shown. c, Arrangement of α6 and α5′ of the PdeL dimer in the R- (PDB: 4LYK) and T-state (PDB: 4LJ3). Residues of the active site and residues involved in the stabilization of the T-state conformation are highlighted and shown as a stick representation. d, Arrangement of α6 and α5′ of the BifA dimer in the R- (PDB: 8ARV) and modelled T-state. Residues of the active site and conserved residues postulated to stabilize the T-state conformation are highlighted and shown as a stick representation. e,f, Standard deviation of the mean of the attachment data shown in Fig. 5d,e, respectively.
Supplementary information
Supplementary Table 1
SCV screen
Supplementary Table 2
Yeast-two-hybrid screen
Supplementary Table 3
SCV suppressor screen
Supplementary Table 4
Structure data collection and refinement statistics
Supplementary Video 1
P. aeruginosa cells expressing hecE remain attached after biofilm dispersal.
Supplementary Video 2
Cell dispersal from flow chamber-grown biofilms is a highly coordinated process.
Supplementary Video 3
Mutants lacking HecE fail to maintain a mature biofilm after cell dispersal.
Supplementary Video 4
Mutants lacking BifA undergo premature biofilm formation and cell dispersal.
Supplementary Video 5
Mutants lacking WspR fail to maintain a mature biofilm after cell dispersal.
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Manner, C., Dias Teixeira, R., Saha, D. et al. A genetic switch controls Pseudomonas aeruginosa surface colonization. Nat Microbiol 8, 1520–1533 (2023). https://doi.org/10.1038/s41564-023-01403-0
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DOI: https://doi.org/10.1038/s41564-023-01403-0
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