Figure 4. Functional investigation of marker gene pdeI in static E. coli biofilm.

(A, B) Uniform Manifold Approximation and Projection (UMAP) plots showing the distribution of pdeI in single-cell data of exponential period E. coli (A) and static E. coli biofilm (B). Each dot represents a cell colored by normalized expression levels of genes. (C) Subcellular localization of PdeI-GFP and GFP. Scale bar, 1 μm. (D) c-di-GMP levels (R^–1 score) in E. coli cells with different BFP, PdeI-BFP, PdeI(G412S)-BFP expression levels (low or high), under the control of the pdeI native promoter, in static E. coli biofilm. c-di-GMP levels are measured using the c-di-GMP sensor system integrated into E. coli cells. R^–1 score was determined using the fluorescent intensity of mVenusNB and mScarlet-I in the system. The fluorescent intensity is measured by flow cytometry (n>50). (E) Determination of cellular concentrations of c-di-GMP by high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in cells overexpressing PdeI under the control of arabinose promoter, with 0.002% arabinose induction for 2 hr (n=3). (F, G) Localization of PdeI-high cells in the biofilm matrix. Cells expressing PdeI-BFP under the control of the pdeI native promoter were grown in a glass-bottom cell culture dish and stained with SYTO 24 for bacterial DNA. Cells expressing BFP under the control of arabinose promoter, with 0.00001% arabinose induction for 24 hr in a glass-bottom cell culture dish and stained with SYTO 24 for bacterial DNA. (H, I) Heterogeneous expression of PdeI in single-cell data of exponential period E. coli (H) and E. coli in static E. coli biofilm (E. coli 24 hr static culture) (I). Biofilm cells with high or low expression levels of PdeI-BFP were sorted by flow cytometry. (J) Persister counting assay using 150 μg/ml ampicillin on cells with high or low expression levels of BFP, PdeI-BFP, and PdeI(G412S)-BFP from static E. coli biofilm, sorted by flow cytometry (n=3). These strains were under the control of the pdeI native promoter. (K) Time-lapse images of the persister assay observed under a microscope. Static biofilm cells of the PdeI-GFP strain were spotted on a gel pad and treated with 150 μg/ml ampicillin in Luria broth (LB). Images were captured over 6 hr at 37°C, followed by the replacement of fresh LB to allow persister cell resuscitation. Scale bar, 2 μm. Error bars represent standard deviations of biological replicates. Significance was ascertained by unpaired Student’s t-test. Statistical significance is denoted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Figure 4—figure supplement 1. Schematic chart for the structure of E. coli PdeI.