Bacterial strains and growth conditions
Bacterial strains used in this study included Escherichia coli strains MG1655, BW25113, UPEC16, and the Keio library Knockout Collection. UPEC16 was isolated from a local UTI patient urine sample (#16) at First Affiliated Hospital of Kunming Medical University following the instruction of the Committee on Human Subject Research and Ethics (2022-L-203 and CHSRE2022030). Additionally, Pseudomonas aeruginosa strain PAO1 and Staphylococcus aureus strain ATCC 25923 were included. Cultures of all strains were grown in Luria Broth (LB) medium. All bacterial strains were routinely grown at 37°C and 220 rpm. To maintain plasmids, when necessary, media were supplemented with the following antibiotics: chloramphenicol (25 µg/ml), kanamycin (20 µg/ml), gentamicin (20 µg/ml), and ampicillin (100 µg/ml). For inducible expression experiments, the following inducers were supplemented in the medium: 0.002%-0.2% arabinose for the Arabinose-induction system, 1 mM IPTG for the Lac-induction system, and 1 µg/ml anhydrotetracycline for the Lac-induction system.
Strains construction
Construction of Target DNA Knockout. Target DNA knockout strains were generated using λ-red mediated gene replacement. The procedure involved the fusion of a 500 bp upstream region, a resistance cassette (CmR for UPEC16 ΔhipH or KanR for UPEC16 ΔpdeI), and a 500 bp downstream region through fusion PCR. The resulting fusion fragments were then transformed into electrocompetent cells along with an induced recombineering helper plasmid, pSim6. After a recovery period of 3–5 hours, the transformed cells were plated on selection plates containing either chloramphenicol or kanamycin. Recombinants were subsequently purified, and their genotypes were confirmed through PCR analysis and sequencing. Finally, mutant strains were incubated at 42°C to eliminate pSim6, which is temperature sensitive.
Recombinant Plasmid Construction. The construction of recombinant plasmids was performed using the 2 × MultiF Seamless Assembly Mix (ABclonal, RK21020). To label the PdeI gene, pdeI, along with its native promoter, was fused with either gfp or bfp and cloned into the pBAD backbone. For gene overexpression, the target genes of interest were cloned into either a p15A ori plasmid or a pUC ori plasmid, under the control of the Arabinose-induction system, Lac-induction system, or Tetracycline induction system. For Gam-GFP overexpression, the gam gene, obtained from bacteriophage Mu, was fused with gfp and cloned into a p15A ori plasmid, under the control of the Tetracycline induction system (Snapgene, pFD152, #125546). Similarly, for recG overexpression, the recG gene was cloned into a p15A ori plasmid, regulated by the T5 promoter-Lac-induction system. To express HipH-His, mCherry-His, DosC, and DgcZ (DgcZ-m), these genes were cloned into pBAD, under the control of the Arabinose-induction system. Additionally, for the c-di-GMP sensor (Snapgene: #182291), the plasmid origin was replaced with the p15A ori.
RiboD PETRI
Cell preparation. E. coli MG1655 cells were grown overnight and then diluted 1:100 into fresh LB medium. After 24 h static growth at 37°C, the culture was vigorously shaken using a vortex, and the cells were pelleted at 5,000g for 2 min at 4°C. The pellet was resuspended in 2 ml of ice-cold 4% formaldehyde (F8775, Millipore Sigma, diluted into PBS). This suspension was rotated at 4°C for 16 h.
Cell permeabilization. 1 ml of fixed cells were centrifuged at 5,000g for 5 min at 4°C and then resuspended in 1 ml washing buffer (100 mM Tris-HCl pH7.0, 0.02 U/µl SUPERase-In RNase Inhibitor, AM2696, Invitrogen). This suspension was centrifuged again at 5,000g for 5 min at 4°C, and the supernatant was removed. The pellet was resuspended in 250 µl permeabilization buffer (0.04% Tween-20 in PBS-RI, PBS with 0.01 U/µl SUPERase-In RNase Inhibitor) and incubated on ice for 3 min. 1 ml cold PBS-RI was added. Then cells were centrifuged at 5,000g for 5 min at 4°C and resuspended in 250 µl Lysozyme Mix (250 µg/ml Lysozyme or 5 µg/ml Lysostaphin for S. aureus) dissolve in TEL-RI buffer: 100 mM Tris, pH 8.0 (AM9856, Invitrogen), 50 mM EDTA (AM9261, Invitrogen), 0.1 U/µl SUPERase In RNase Inhibitor). Samples were incubated at 37°C and mixed gently per min. Then 1ml cold PBS-RI was added immediately and cells were centrifuged at 5,000g for 5 min at 4°C. Cells were washed again using 1 ml cold PBS-RI. Cells were resuspended in 40 µl DNaseI-RI buffer (4.4 µl 10×reaction buffer, 0.2 µl SUPERase In RNase inhibitor, 35.4 µl H2O) and 4 µl DNaseI (AMPD1, Millipore Sigma) was added. Samples were incubated for 30 min at room temperature and mixed gently per 5 min. 4 µl Stop Solution was added and samples were incubated for 10 min at 50°C and mixed gently per min. Then cells were centrifuged at 5,000g for 10 min at 4°C and washed twice using 0.5 ml cold PBS-RI. Cells were resuspended in 200 µl cold PBS-RI. The cells were counted using ACEA NovoCyte flow cytometer and checked cell integrity using 100×oil immersion lens.
Primers preparation. For round 1 RT, round 2 and round 3 ligation reactions, all primers design and preparation as previously described (15). All primers were purchased from Sangon Biotech (table S1). For ligation primers preparation, mixtures were prepared as follows: 31.1 µl each R2 primer (100 µM), 28.5 µl SB83 (100 µM) and 21.4 µl H2O, were splitted to 2.24 µl for one sample. Mixtures containing 63.2 µl each R3 primer (70 µM) and 58 µl SB8 (70 µM), were splitted to 3.49 µl for one sample. Splitted ligation primers could be saved at -20°C. Before use, ligation primers were incubated as follows: 95°C for 3 min, then decreasing the temperature to 20°C at a ramp speed of − 0.1°C s − 1, 37°C for 30 min. For blocking mix preparation, 50 µl primer SB84 (400 µM) and 80 µl primer SB81 (400 µM) were incubated as follows: 94°C for 3 min, then decreasing the temperature to 25°C at a ramp speed of − 0.1°C s − 1, 4°C for keeping. Round 2 blocking primers were mixed as follows: 37.5 µl 400 µM SB84, 37.5 µl 400 µM SB85, 25 µl 10× T4 ligase buffer, 150 µl H2O. Round 3 blocking primers were mixed as follows: 72 µl 400 µM SB81, 72 µl 400 µM SB82, 120 µl 10× T4 ligase buffer, 336 µl H2O, 600 µL 0.5 M EDTA.
Round 1 RT reactions. About 3×107 cells were added to a RT reaction mix consisting of 240 µl 5× RT buffer, 24 µl dNTPs (N0447L, NEB), 12 µl SUPERase In RNase Inhibitor and 24 µl Maxima H Minus Reverse Transcriptase (EP0753, Thermo Fisher Scientific). Nuclease Free water was added to bring the volume of the reaction mixture to 960 µl and this reaction solution should be mixed thoroughly by vortex. 8 µl reaction mixture was added to each well of the 96-well plate, in which 2 µl each RT primer was added previously. The 96-well plate was sealed and mixed reaction by turn upside down repeatedly. Following by short spin, the 96-well plate was incubated as follows: 50°C for 10 min, 8°C for 12 s, 15°C for 45 s, 20°C for 45 s, 30°C for 30 s, 42°C for 6 min, 50°C for 16 min and then held at 4°C. After RT, all the 96 reactions were pooled into one tube. 75 µl 0.5% Tween-20 was added and the reactions were incubated on ice for 3 min. Cells were centrifuged at 7,000g for 10 min at 4°C and resuspended in 0.4 ml PBS-RI. 32 µl 0.5% Tween-20 was added and cells were centrifuged at 7,000g for 10 min at 4°C.
Round 2 ligation reactions. Cells were resuspended in 500 µl 1×T4 ligase buffer and then 107.5 µl PEG8000, 37.5 µl 10×T4 ligase buffer, 16.7 µl SUPERase In RNase Inhibitor, 5.6 µl BSA, and 27.9 µl T4 ligase (M0202L, NEB) were added in. This reaction solution should be mixed thoroughly by vortex. 5.76 µl reaction mixture was added to each well of the 96-well plate, in which 2.24 µl each round 2 ligation primer was added in previously. The 96-well plate was sealed and mixed reaction by turn upside down repeatedly. Following by short spin, the 96-well plate was incubated at 37°C for 45 min. Then, 2 µl of round 2 blocking mix was added to each well and incubated at 37°C for 45 min. All the 96 reactions were pooled into one tube.
Round3 ligation reactions. 89 µl H2O, 26 µl PEG8000, 46 µl 10 × T4 ligase buffer, and 12.65 µl T4 ligase were mixed together. This reaction solution should be mixed thoroughly by vortex. 8.51 µl reaction mixture was added to each well of the 96-well plate, in which 3.49 µl each round 3 ligation primer was added previously. The 96-well plate was sealed and mixed reaction by turn upside down repeatedly. Following by short spin, the 96-well plate was incubated 37°C for 45 min. Then, 10 µl of round 3 blocking mix was added to each well and incubated 37°C for 45 min. All the 96 reactions were pooled into one tube.
Cells lysis. 42 µl 0.5% Tween-20 was added and cells were centrifuged at 7,000g for 10 min at 4°C. Cells were washed twice using 200 µl TEL-RI containing 0.01% Tween-20 at 7,000g for 10 min at 4°C. Then cells were resuspended in 30 µl TEL-RI buffer. Cells were counted using ACEA NovoCyte flow cytometer and checked cell integrity using 100×oil immersion lens. Moderate amounts of cells were added in lysis buffer (50 mM Tris pH 8.0, 25 mM EDTA, 200 mM NaCl, 0.5% Triton X-100) and then 5 µl proteinase K (AM2548, Invitrogen) were added. Samples were incubated 55°C for 60 min and and mixed gently per min.
Library construction. There are many modifications, especially r-cDNA depletion. For template switch, Lysates were purified with VAHTS DNA Clean Beads (N411, Vazyme) at a ratio of 2.0× and cDNA was eluted in 12 µl of water. 4 µl 5× RT buffer, 1 µl dNTPs (N0447L, NEB), 0.5 µl SUPERase In RNase Inhibitor, 0.5 µl Maxima H Minus Reverse Transcriptase and 0.5 µl TSO primer (100 mM, table S1) were added to the purified cDNA. This reaction solution was incubated as follows: 25°C for 30 min, 42°C for 90 min, 85°C for 5 min and then held at 4°C. 1 µl RNaseH were added and this reaction solution was incubated 37°C for 30 min. cDNA were purified with VAHTS DNA Clean Beads at a ratio of 2.0× and eluted in 13 µl of H2O. cDNA integrity was checked using primers TSO-2 and R1 or R2 or R3 by qPCR (table S1).
Ribosome RNA derived cDNA depletion (RiboD): we designed a series of cDNA probe primers to deplete r-cDNA (table. S1). These probe primers could hybridize with r-cDNA specifically and also hybridize with a biotin-labeled universal primer. 5 µl r-cDNA probe primers (10 µM), 2.5 µl 10× hybridization buffer (Tris-HCl pH8.0 100 mM, NaCl 500 mM, EDTA pH8.0 10 mM) and 5 µl biotin primer (10 µM) were added to 12.5 µl purified cDNA. This reaction solution was incubated as follows: 95°C for 2 min, then decreasing the temperature to 20°C at a ramp speed of − 0.1°C s − 1, 37°C for 30 min. Aliquots of 20 µl Streptavidin magnetic beads (BEAVER, 22307) were washed twice using 1 ml 1× B&W buffer (Tris-HCl pH7.5 10 mM, EDTA 1 mM, NaCl 1M, Tween-20 0.05%) and resuspended in 25 µl 2× B&W buffer. 25 µl washed Streptavidin magnetic beads were added to 25 µl annealed cDNA. This reaction solution was incubated room temperature for 30 min and mixed per min. Place the reaction solution tube into a magnetic stand to collect the supernatant. cDNA depleted r-cDNA were purified with VAHTS DNA Clean Beads at a ratio of 2.0× and eluted in 12.5 µl of H2O. r-cDNA could be deplete again using above protocol and finally cDNA was eluted in 20 µl of H2O.
Library amplification and sequencing. 2.4 µl R3 primer (10 mM, table S1), 2.4 µl TSO-2 primer (10 mM, table S1), 40 µl 2× KAPA HIFI mix (KAPA, 2602), 1.6 µl Sybr green (25×), 0.8 µl MgCl2 (0.1 M) and 12.8 µl H2O were added to 20 µl cDNA. This PCR reaction solution was placed in thermocycler and incubated as follows: 98°C for 45 s, beginning cycling of 98°C for 15 s, 60°C for 30 s, 72°C for 60 s. Cycles were allowed to continue on a qPCR machine until reaction neared saturation. PCR products were purified with VAHTS DNA Clean Beads at a ratio of 0.9× and eluted in 25 µl of H2O. Finally, Purified PCR products were end repaired and ligated adaptor using VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607).
Bulk RNA-seq library construction
The total RNA of the samples was extracted using the Bacteria RNA Extraction Kit (R403-01, Vazyme) and subjected to mRNA selection (N407, Vazyme), fragmentation, cDNA synthesis, and library preparation using the VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina® (NR603, Vazyme).
Bioinformatics analysis Methods
Single-Cell Analysis. The sequencing data was processed into a matrix by scripts and pipeline as previously described (15) in Python 2.7.15 with some modifications (detailed original code deposited to GEO repository). Downstream analysis of single cell data was performed in Seurat v4.3.0. Data was first preprocessed filtering out cells that mapped genes more than 100 and less than 2000 for replicate 1, or more than 150 and less than 2000 for replicate 2. The data was then normalized by a scale factor of 10000 by employing a global-scaling normalization method “LogNormalize”. Highly variable features were identified and return 500 features per dataset. The data was then scaled by the ScaleData function, and dimension was reduced using principal component analysis. Following principal component analysis, we performed graph-based clustering approach to identify clusters of gene expression programs using the Louvain algorithm (Seurat 4.3.0). Marker genes for each cluster were computed using the Wilcoxon Rank-sum test. Briefly, marker genes for each cluster were first obtained using the FindMarkers function in Seurat.
Comparison of scRNA-seq with Bulk RNA-Seq. Raw reads from the bulk data were aligned to the E. coli MG1655 k12 genome and annotated as described above. Single-cell sequencing data were combined with the gene expression numbers of all cells according to the bulk RNA-seq analysis methods. Single-cell and bulk transcriptomes of E. coli were compared by computing the Pearson correlation of log2 reads of each gene between the two measurements.
Chip-seq Analysis. High-throughput sequencing of Chip-seq library was performed on the Genome Analyzer IIx (Illumina). For Chip-Seq analysis, the quality of the raw data of the prepared libraries were assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were then mapped to the MG1655 k12 genome using the BWA aligner software (https://github.com/lh3/bwa.git). Then we converted the sam files to bam files using samtool 1.9. The bam files were then analysed using macs2 (https://github.com/taoliu/MACS/) to find enriched regions of reads for the ChIP-seq data. And ChIPseeker was used to calculates and visualises the area covered by the peak on the chromosome through the covplot function with files in narrow.peak format. For motif discovery, MEME (https://meme-suite.org/meme/) was used to find binding motif enriched in the sequencing data.
gDNA library screening
gDNA library construction was performed as described previously (36) with some major modifications. The gDNA of MG1655 Δara was digested with DNaseI in Mg2+ buffer. 3000–6000 bp fragments were purified from agarose gel and end-repaired via Fast DNA end repair Kit (Thermo, K0771). These fragments were inserted into linearized pUC19 which was digested with SmaI and then dephosphorylated through T4 DNA ligation reaction. gDNA library contained more than 20,000 recombinant plasmids. This library was transformed into MG1655 Δara barboring p15G::hipH and plated on LB plates after arabinose induction.
Persister assay
Strains culture, including mid-exponential and proteins overexpression, was diluted by 1:20 into fresh LB broth containing 150 µg/ml ampicillin and incubated again for 3 h at 37°C and 220 rpm. For the cells, sorted from FACS or harvested from static biofilm or mice bladder, were resuspend in fresh LB broth containing 150 µg/ml ampicillin and incubated again for 3 h at 37°C and 220 rpm. Cells were plated on LB plate for colony-forming unit (CFU) counts before ampicillin challenge incubated overnight at 37°C. After ampicillin challenge, Cells were washed with PBS buffer and plated for CFU counts again. Persister ratio means the ratio of the CFU after ampicillin challenge to the CFU before ampicillin challenge. For HipH overexpression sample, persister ratio means the ratio of the CFU after ampicillin challenge to the CFU before arabinose induction. Averages and standard deviations are representative of three biological replicates.
Cell growth potential assay and VBNC determination.
Strains harboring plasmid to overexpression toxin HipH under the control of arabinose-induction system were grown to exponential phase in LB medium. Then HipH was induced by 0.002% arabinose for 3 to 4 h at 37°C and 220 rpm. Cells were plated for CFU counts on LB plate before and after arabinose induction. Cells with growth potential is the ratio of the CFU after arabinose induction to the CFU before arabinose induction. Cells overexpressed HipH were stained by PI dye to count dead cells ratio. The ratio of VBNC is 100% minus the ratio of cells with growth potential and dead cells. Averages and standard deviations are representative of three biological replicates.
Microscopy
Bright-field and fluorescence imaging. Inverted microscope (Nikon, eclipse Ti2 and Leica, Stellaris 5 WLL) were used. The illumination was provided by different lasers, at wavelengths 405 nm for BFP and Hoechst 33258, 488 nm for GFP, 488 nm (Nikon) or 515 nm (Leica) for mVenusNB, 561 nm for propidium iodide (PI) and mScarlet, respectively. The fluorescence emission signal was imaged to an sCMOS camera (pco.edge 4.2 bi). Appropriate filter sets were selected for each fluorophore according to their spectrum. Image analysis was done by ImageJ software (Fiji). For c-di-GMP sensor analysis, the ratio of mVenusNB to mScarlet-I (R) is negative correlation to the concentration of c-di-GMP. So, R− 1 is positive correlation to the concentration of c-di-GMP.
Cells staining. PI, Hoechst 33258 and SYTOTM24 were added to culture at a final concentration of 1 µg/ml, 10 µg/ml and 10 µM, respectively. The cells were incubated for 15 to 30 min in dark at room temperature. Before imaging, cells were washed twice using PBS.
DNA diffusion assay. Cells were prepared as previously described with some modifications (32, 33). About 2×106 E. coli cells overexpressed HipH in 20 µl PBS were mixed with 60 µl melted 0.7% low melting agarose. This mixture was pipetted on to a precoated slide which was coated with 1% agarose and dried at 80°C for 10 min. This slide was covered with a coverslip and incubated for 10 min at 4°C. The coverslip was gently removed, and the slide was horizontally immersed in 10 ml of preheated lysis solution (2% SDS, 0.05 M EDTA, and 0.1 M DTT, pH 11.5) for 5 min in a closed tray at 37°C. Then the slide was washed horizontally in a tray with abundant distilled water for 3 min at room temperature. After air-drying, the slide was dehydrated horizontally by in cold ethanol baths sequentially (70, 90, and 100%, for 3 min each). The slide was stained with PI (30 µM) for 10 min in dark and visualized under a fluorescence microscope.
Gam-GFP foci imaging is modified from the previous description (34), strains overexpressing toxin HipH along with DosC or DgcZ under the arabinose-induction system and overexpressing Gam-GFP under Tetracycline-induction system grew to exponential phase in LB medium. Gam-GFP was induced by 1 mg/ml anhydrotetracycline for 30 min and then HipH and DosC (or DgcZ) were induced by 0.2% arabinose for 2.5 h at 37°C and 220 rpm. Cells were washed two times using PBS buffer and then visualized under a fluorescence microscope. The Gam-GFP foci were analyzed by ImageJ software. From the resulting individual cell intensity histograms, we consider pixel fluorescence intensity (f) that were above the mean fluorescent intensity of the cell (a). We designated these pixels as foci if their intensity (f) was greater than 1.2 times the mean intensity (1.2a) and if at least four such pixels were found to be adjacent to each other. Additionally, to qualify as foci, the area occupied by these pixels needed to be less than one-fifth of the total bacterial area.
Time-lapse imaging. For recording antibiotic killing and bacteria resuscitation processes, cells labelled by PdeI-GFP or harboring c-di-GMP sensor in the 24 h static growth phase were collected and washed twice with PBS buffer and imaged on a gel-pad containing 3% low melting temperature agarose in PBS (27, 37). The gel-pad was prepared in the center of the FCS3 chamber as a gel island. The cells were observed under bright field or epifluorescence illumination. Then, the gel-pad was surrounded by LB broth containing 150 µg/ml ampicillin for 6 h at 35°C. Fresh LB broth was flushed in, and the growth medium was refreshed every 3 h, allowing cells to recover sufficiently.
Flow cytometry and FACS analysis
All samples were measured on a Beckman CytoFLEX SRT flow cytometer with a 70 µm nozzle in which normal saline was used as sheath fluid. PdeI-BFP or c-di-GMP sensor labeled strain in the 24 h static biofilm growth phase was washed and resuspended in sterile PBS. Microorganisms were identified by FSC (forward scatter) and SSC (side scatter) parameters. Cells were sorted into different groups based on their fluorescence intensity (PB450 for BFP, FITC for mVenusNB, ECD for mScarlet-I). The results were analyzed by FlowJo V10 software (Treestar, Inc.).
Chip-seq and motif analysis
gDNA library construction was performed as described previously (38) with major modifications. MG1655 Δara harboring pBAD::hipH-Flag grew to mid-exponential growth phase in 250 ml LB medium. HipH-Flag were induced by 0.002% arabinose for 2 h at 37°C and 220 rpm. 1% formaldehyde were added in for 0.5 h at 37°C and 0.38 M Glycine were added in to stop the crosslinking reaction. Cells were harvested and washed by TBS buffer. Cells were divided to six fractions. Each fraction was resuspended in 5 ml SMM buffer (0.02 M maleic acid, 0.5 M sucrose, 0.02 M MgCl2, pH 6.5) supplemented with 5 mg/ml lysozyme and 1 mM PMSF and incubated at 37°C for 20 min with mixing. Cells were pelleted and washed by SMM buffer. The pellets were resuspended in 400 µl of King2 buffer (0.1 M Tris-Cl, pH 7.5, 0.2 M NaCl, 1% Triton X-100, 0.1% Na-deoxycholate, 0.2% Brij 58, 20% glycerol) containing 1×protease inhibitor cocktail and 0.25 mg/ml RNaseA. 50 µl Mg-Ca solution (final concentration: 10 mM MgCl2, 5 mM CaCl2) and 50 µl of DNaseI (final concentration: 1 U/ml) were added in. This reaction mix was incubated at 37°C for 30 min. 3 mL of UT buffer (0.1 M HEPES, 8 M Urea, 0.5 M NaCl, 1% Triton X-100, 10 mM β-mercaptoethanol) containing 1 mM PMSF were added in. Cells were ruptured by sonication. Each cells lysate was centrifuged at 12,000g for 10 min at 4°C and supernatant was collected for immunoprecipitation. 4 µg/ml antibody of Flag (MBL, M185-3L) were added in each sample in which two samples containing 20 µM C-di-Gmp. 4 µg/ml antibody of IgG control (Proteintech, 66360) were added in another two samples. All samples were incubated at 4°C for overnight. Then 100 µl washed A/G Beads (BeaverBeads™, 22202) were added in. All samples were incubated at 4°C for 4 h. A/G Beads were washed five times by TBST buffer and then eluted by M-wash-buffer (0.1 M Tris–HCl, pH 7.5, 1% SDS, 10 mM DTT). Elution samples were incubated at 65°C for 15 h. All DNA fragments were purified by Phenol-chloroform method. Finally, purified DNA fragments were constructed DNA library for sequencing by VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607).
Protein purification
All proteins used in this study were purified by affinity chromatography of His-tag and nickel column. Briefly, MG1655 Δara harboring pBAD::hipH-GFP-His (or GFP-His) grew to exponential growth phase in 500 ml medium. HipH-GFP-His (or GFP-His) were induced by 0.002% arabinose for 2 h at 37°C and 220 rpm. Cells were harvested and resuspended in 25 ml binding buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM imidazole, 0.5% Triton X-100, pH 8.0) containing 1 mg/ml lysozyme and 5 µg/ml DNaseI. Samples were incubated at 37°C for 30 min and then frozen and thawed three times. Cells lysate were centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected for proteins purification. Ni-TED SefinoseTM Resin (Sangon Biotech, C610030) were used to purify His tagged proteins followed by Manual. Finally, HipH-GFP-His (or GFP-His) were storage in elution buffer containing 10% glycerol at -80°C.
Determination of c-di-GMP concentration by HPLC-MS/MS
MG1655 Δara pBAD::pdeI, or dosC, or dgcZ were grown to mid-exponential growth phase and then induced by 0.002% arabinose. After 2 h incubation, cells were harvested and washed by PBS. The washed cells were quickly frozen by liquid nitrogen. At the same time, another part of washed cells was stained by SYTOTM24 and counted by Flow cytometry. C-di-GMP concentration detection were performed by WUHAN Lixinheng technology Co. Ltd. via high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All strains were performed in biological triplicates. Measured values were converted into intracellular C-di-GMP concentrations (pg) per cell.
Biotin c-di-GMP pull down assay
MG1655 Δara harboring pBAD::hipH-His (or mCherry-His) grew to mid-exponential growth phase in 30 ml medium. HipH-His (or mCherry-His) were induced by 0.002% arabinose for 2 h at 37°C and 220 rpm. Cells were harvested and resuspended in 1.5 ml lysis buffer (TBST containing 1 mg/ml lysozyme, 5 µg/ml DNaseI, 1×protease inhibitor). Samples were incubated at 37°C for 30 min and then frozen and thawed three times. Cells lysate were centrifuged at 12,000g for 20 min at 4°C. The supernatant was collected for pull down assay. The pull down assay was as previously described (39) with some modifications. Indicated amount of lysate was added into reaction mix containing indicated amount of biotin-c-di-GMP (200 pmol/µl, BIOLOG, B098), 1 µl EDTA (200 mM), 10 µl 10×buffer (100 mM Tris-HCl, pH 7.5, 500 mM KCl, 10 mM DTT). H2O were added into reaction mix up to 100 µl. This reaction mix was incubated at 25°C for 20 min. 10 µl (per sample) Streptavidin (SA) magnetic beads (Thermo Scientific, 88816) was washed twice using TBST and resuspended in 150 µl TBST. Washed SA beads were added into reaction buffer and incubated at 25°C for 1 h on a rotary table. SA beads then were washed five times using TBST. Biotin-c-di-GMP conjugated proteins on SA beads were eluted by SDS loading buffer and analyzed by Western blot.
DNA cleavage assay
For HipH binding C-di-Gmp, 1 µl purified HipH-GFP-His protein (6 µM) or GFP-His protein (6 µM) was added into binding buffer containing 2 µl 10 × buffer (100 mM Tris-HCl, pH 7.5, 500 mM KCl, 10 mM DTT), 0.2 µl EDTA (200 mM), 0 µl to 15 µl c-di-GMP (10 mM). H2O were added into the reaction mix up to 20 µl. This binding mix was incubated at 30°C for 1 h. For DNA cleavage, 500 ng supercoiled pBR322 collected from FastPure Plasmid Mini Kit (Vazyme, DC201-01) or relaxed pBR322 which were from supercoiled pBR322 digested by TopoisomeraseI (Takara, 2240A), 6 µl 5×buffer (170 mM Tris-HCl, pH 7.5, 120 mM KCl, 10 mM DTT, 20 mM MgCl, 25 mM spermidine, 25% Glycerol) and 3 µl BSA (1 mg/ml) were added into binding mix. H2O were added into reaction mix up to 30 µl. This reaction mix was incubated at 37°C for 2 h. The reactions were stopped by the addition of SDS to a final concentration of 0.2% and incubated at 37°C for 30 min. Product DNA was analyzed by 1% (w/v) agarose gel electrophoresis.
Mouse bladder infection
UPEC16 and its mutant strains, ΔpdeI and ΔhipH, grew to mid-exponential growth phase. Cells were harvested and washed by PBS. Cell pellets were resuspended in PBS to infect mice. Bladder infection was performed as described previously with minor modifications (40). 8-week-old BALB/c mice were anesthetized by isoflurane. 2*108 CFU indicated strains were transurethrally inoculated by use of 25G blunt needle. At 24 h, mice were infection again. At 48 h, mice were humanely killed by cervical dislocation and the bladder was aseptically removed and homogenized in 1 mL of sterile PBS. Homogenates were filtered through 70 µm Cell filters and then plated on LB plate for CFU counts and persister detection. Animal studies were approved by the Animal Care Ethics Committee of Medical Research Institute, Wuhan University. We confirm that all experiments conform to the relevant regulatory standards.
Statistical analysis
Statistical analysis was performed in GraphPad Prism 9 software for Windows. Significance was ascertained by two-tailed Student’s t test. Error bars represent the standard deviations of the mean from at least three independent experiments. A value of P < 0.05 was considered significant. “ * ” indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).