Ethics statement

All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. The protocol was approved by the Animal Welfare and Research Ethics Committee of Northwest A&F University (protocol number: NWAFUSM2018001). Six-week-old female mice (BALB/c) were purchased from the central animal laboratory of Xi’An JiaoTong University (Xi’an, China) and kept in a temperature (24 ± 2 °C), 50 ± 10% humidity, air flow of 35 exchanges and light-controlled room (12 h light, 12 h darkness) with free access to food and water.

Bacteria strains and growth conditions

Bacteria strains and plasmids used in this study are listed in Supplementary Table 1. Yptb strains were grown in YLB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) or M9 medium (Na₂HPO₄, 6 g L⁻¹; KH₂PO₄, 3 g L⁻¹; NaCl, 0.5 g L⁻¹; NH₄Cl, 1 g L⁻¹; MgSO₄, 1 mM; CaCl₂, 0.1 mM; glucose 0.2%) at 26 °C or 30 °C. E. coli and S. Typhimurium were cultured in LB broth at 37 °C or 26 °C. Appropriate antibiotics were included in growth medium and their corresponding concentrations are: Ampicillin (100 μg ml⁻¹), Nalidixic acid (20 μg ml⁻¹), Kanamycin (50 μg ml⁻¹), Tetracycline (5 μg ml⁻¹ for Yptb and 15 μg ml⁻¹ for E. coli), Gentamicin (20 μg ml⁻¹), Chloramphenicol (20 μg ml⁻¹).

Plasmid construction

Primers used in this study are listed in Supplementary Table 2. For obtaining expression plasmids, the genes encoding Yptb Tce1 (YPK_0954) was amplified by PCR. The DNA fragment was digested and cloned into similarly digested pGEX6p-1 and pET28a vectors, yielding corresponding plasmid derivatives. The expression clones of Tci1 (YPK_0955), BtuB (YPK_0782), OmpF (YPK_2649), TolB (YPK_2956) were obtained with the same method. As for the expression plasmid pET28a-tce1-gfp, primers tce1-F-BamHI and tce1-R-EcoRI (TAA) were used to amplify tce1 without termination codon TAA, and GFP-F-EcoRI and GFP-R-SalI were used to amplify the gfp fragment. Digested fragments were inserted into pET28a to produce pET28a-tce1-gfp. The plasmid pDM4-Δtce1 used to construct the Δtce1 in-frame deletion mutant was made by overlap PCR. Briefly, the 800-bp upstream fragment and the 800-bp downstream fragment of tce1 were amplified with primer pairs tce1-M1F-BamHI/tce1-M1R and tce1-M2F/tce1-M2R-SalI, respectively. The upstream and downstream PCR fragments were fused with the primer pair tce1-M1F-BamHI/tce1-M2R-SalI by overlap PCR. The resulting PCR products were digested with BamHI and SalI, and inserted into similar digested suicide plasmid pDM4 to produce pDM4-Δtce1. The knock-out plasmid pDM4-Δtci1, pDM4-ΔbtuB, pDM4-ΔompF, pDM4-Δypk_2801-2802 were constructed with similar manners by using primers listed in Supplementary Table 2. To construct plasmids used in bacterial two-hybrid complementation assays, the genes for testing were amplified by PCR from Yptb genomic DNA using appropriate primers. Amplified DNA fragments were digested with appropriate restriction enzymes, and cloned into the corresponding sites of pKT25 and pUT18C vectors, respectively. The plasmid pTargetF1-ΔbtuB_Ec was used to construct the ΔbtuB_Ec in-frame deletion mutant in E. coli DH5α. To construct pTargetF1-ΔbtuB, upstream and downstream of gene btuB_Ec were amplified by PCR using primer pairs B3966-up-F/B3966-up-R and B3966-down-F/B3966-down-R respectively. The DNA fragment that code for btuB_Ec targeted sgRNA was made by PCR against pTargetF1 using primer pair B3966-g20-F/B3966-g20-R. Overlapping PCR was used to combine these three parts in the order of sgRNA-Up-Down and the resulting assembly was ligated to pTargetF1 pre-digested with SpeI/SalI using Gibson Assembly. With a similar method, plasmid pTargetF1-ΔompF_Ec was produced. To complement the Δtce1 mutant, primers tce1-F-BamHI and tce1-R-SalI were employed to amplify the tce1 gene fragment from Yptb genomic DNA. The PCR product was digested with BamHI/SalI and ligated into similarly digested pKT100 to produce pKT100-tce1. The complementary plasmids pKT100-tci1, pKT100-btuB, pKT100-tolB, pKT100-ompF, pKT100-btuB_Ec, and pKT100-ompF_Ec were similarly constructed. Plasmid pME6032-tce1-vsvg was constructed for protein secretion assay. Briefly, primers tce1-F-EcoRI and tce1-R-VSVG-BglII were employed to amplify the tce1 gene from Yptb genomic DNA. The PCR product was digested with EcoRI/BglII and inserted into similarly digested pME6032 to generate pME6032-tce1-vsvg. pME6032-ypk_0952-vsvg and pME6032-tci1 were constructed in the same way. The integrity of the insert in all constructs was confirmed by DNA sequencing.

In-frame deletion and complementation

To construct in-frame deletion mutants, pDM4 derivatives were transformed into Yptb through E. coli S17-1 λpir-mediated conjugational mating, achieved by mixing 50 μl volumes of overnight LB cultures of E. coli S17-1 λpir donor with the Yptb parent. The mixture was spotted onto a non‐selective LB plate and incubated for 16 h at 30 °C for mating. Integration of the introduced plasmid into Yptb chromosome by single cross-over was selected on YLB plates containing 20 μg ml⁻¹ chloramphenicol and 20 μg ml⁻¹ nalidixic acid. The chloramphenicol-resistant colonies were grown overnight in LB broth allowing for a second cross-over to occur. Selection for loss of the genome integrated sacB-containing plasmid was performed on YLB plates containing 20% sucrose and 20 μg ml⁻¹ nalidixic acid. Strains growing on this plate were tested for chloramphenicol sensitivity by parallel spotting on YLB plates containing either chloramphenicol or nalidixic acid. Chloramphenicol-sensitive and sucrose-resistant colonies were tested for deletion by PCR and confirmed by DNA sequencing^39,40. For overexpression or complementation in relevant Yptb strains, the pME6032 or pKT100 derivatives were transformed into relevant Yptb strains by electroporation and the expression in Yptb was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

Clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9) system was used to construct deletion mutants in E. coli DH5α according to the method described previously⁴¹, with the spectinomycin resistance gene in the pTargetF plasmid replaced by a chloramphenicol resistance gene (pTargetF1). The pTargetF1 derivatives harboring sg-upstream-downstream were electroporated in E. coli pre-transferred with vector pCas at 30 °C. The mutant colonies were selected on LB plates containing 50 μg ml⁻¹ kanamycin and 20 μg ml⁻¹ chloramphenicol. The plasmids pTargetF and pCas in the ΔbtuB_Ec and ΔompF_Ec mutant were successively eliminated by IPTG (1 mM) induction at 30 °C and overnight incubation at 37 °C, respectively⁴². After curing of the pTarget series and pCas plasmids, the deletion mutants were in trans complemented with pKT100 carrying their respective genes.

Overexpression and purification of recombinant proteins

To express and purify His₆ and GST-tagged recombinant proteins, pET28a and pGEX6P-1 derivatives were transformed into E. coli BL21(DE3) and E. coli XL-1 Blue, respectively. Bacteria were cultured in 5 ml LB at 37 °C to reach stationary phase and re-inoculated with a ratio of 1:100 into fresh LB, cultivated at 37 °C until OD₆₀₀ = 0.40. Then 0.2–0.5 mM IPTG was added into the growth medium, continue cultivation for another 12 h at 22 °C in a rotary shaker with a speed setting of 150 rpm. Cells were collected and disrupted by sonification and purified with the His•Bind Ni-NTA resin or GST•Bind Resin (Novagen, Madison, WI), respectively, according to manufacturer’s instructions. Purified proteins were dialyzed against PBS (phosphate-buffered saline) at 4 °C overnight. To get highly purified His-tagged Tce1 and Tce1^S8A/A16E proteins, further processes were adopted after His•Bind Ni-NTA resin purification. The eluted samples from Ni-NTA were desalted into QA buffer (20 mM Tris-HCl, 0.2 M NaCl, 10% glycerol, pH 7.5) and loaded onto HiTrap Q HP 1 ml using AKTA Pure 25 chromatography system (GE healthcare, USA). A 20 ml salt concentration gradient from 0.2 to 1 M of NaCl was performed to separate the protein samples. His-tagged Tce1 and Tce1^S8A/A16E proteins were eluted at about 0.35 M NaCl and pooled. The recombinant proteins were over 85% purity analyzed by SDS–PAGE.

To express and purify outer membrane proteins, E. coli BL21(DE3) that contains the corresponding expression vector was grown in 5 ml LB at 37 °C and transferred into 500 ml LB until OD₆₀₀ reached 0.4. 0.3 mM IPTG was added and the growth condition of bacteria was shifted to 22 °C with shaking at 150 rpm. Incubated cells were collected and resuspended in binding buffer (20 mM Tris-HCl, 100 mM glycine, pH 8.3) with 6 M urea, and then it was centrifuged again to remove the residual membranes. The supernatant was purified with the His•Bind Ni-NTA resin and eluted with elution buffer. The denatured protein was mixed with refolding buffer (55 mM Tris-HCl, 0.21 mM NaCl, 0.88 mM KCl, 880 mM L-arginine, 0.5% SB-12, pH 7.0) with the ratio of 1/20 followed by 4 °C overnight incubation. The refolded protein was ultrafiltered to increase its purity and concentration and then dialyzed with buffer containing 55 mM Tris-HCl (pH 6.5), 0.21 mM NaCl, 10 mM l-arginine, and 0.5% SB-12²⁰. Protein concentrations were determined by the Bradford assay with BSA (bovine serum albumin) as standard.

GST pull-down assay

To screen for binding partners of Tce1 with GST pull-down^20,43, 0.5 mg purified GST-Tce1 protein was incubated with 100 μl pre-washed glutathione beads for 2 h at 4 °C, then mixed with cleared cell lysates collected from 100 ml of Yptb culture for another 4 h. After incubation, the beads were collected and washed three times with PBS containing 300 mM NaCl, and three times with PBS containing 500 mM NaCl. Proteins binding on the beads were boiled with SDS sample buffer, resolved by SDS–PAGE and visualized by silver staining (Bio-Rad). Individual protein bands on the gel were excised, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization/mass spectrometry (Voyager-DE STR, Applied Biosystems, Waltham, MA). To analyze protein interactions, purified GST fusion protein was mixed with 6×His fusion protein in PBS on a rotator for 2 h at 4 °C, and GST or an irrelevant protein CheY (BTH_II2365 in Burkholderia thailandensis) fused to GST were used as negative controls. After adding 40 μl of pre-washed glutathione beads slurry, binding was allowed to proceed for another 2 h at 4 °C. The beads were then washed five times with TEN buffer (100 mM Tris-Cl, 10 mM EDTA, 500 mM NaCl, pH 8.0). Retained proteins were resolved by SDS–PAGE and visualized by western blot.

Bacterial two-hybrid assay

To perform bacterial two-hybrid complementation assays^44,45, the pKT25 and pUT18C derivatives were co-transformed into E. coli BTH101 and cultured on MacConkey plate (Ampicillin 100 μg ml⁻¹, Kanamycin 50 μg ml⁻¹, IPTG 1 mM) at 30 °C. At the same time, the plasmid pKT25-zip/pUT18C-zip and pKT25/pUT18C were co-transformed into E. coli BTH101 to serve as positive and negative controls, respectively. Interactions were tested using MacConkey medium and a red colony color shows an interaction between proteins, while a white colony color attests the absence of interaction. Efficiencies of interactions between different proteins were quantified by measuring β-galactosidase activities in liquid cultures. In brief, overnight cultures were diluted to 1% and grown in LB broth with antibiotics at 30 °C until OD₆₀₀ reached 1.0 and β-galactosidase activities were assessed using ONPG as the substrate.

Growth inhibition assay

E. coli BL21(DE3) harboring pET28a empty vector, pET28a-tce1, pET28a-tce1^S8A/A16E, and pET28a-tce1-tci1 were grown in LB medium. Overnight cultures were adjusted to the same OD₆₀₀ value and diluted 100-fold into LB broth containing appropriate antibiotics. After incubated at 26 °C, 180 rpm for 2 h, the expression of recombinant proteins was induced by the addition of 0.5 mM IPTG, and incubated continually under the same condition. The growth of cultures was monitored by measuring OD₆₀₀ at 2 h intervals.

Protein toxicity assay

Stationary-phase bacteria strains grown in YLB medium were collected, washed and diluted 40-fold into M9 medium, and treated with purified Tce1 and Tse1 toxins (0.005, 0.01, or 0.1 mg ml^–1) at 30 °C with shaking at 100 rpm for 60 min. After treatment, the cultures were serially diluted and plated onto YLB agar plates, and colonies were counted after 36 h growth at 30 °C. Percentage survival was calculated by dividing the number of CFU of treated cells by the number of CFU of cells without toxin treatment. All these assays were performed in triplicate at least three times.

Protein secretion assay

To perform protein secretion assays⁴⁶, Yptb strains were grown in 3 ml YLB at 30 °C and transferred into 300 ml M9 medium with 1 mM IPTG until OD₆₀₀ reached 0.60–0.65. Two milliliter of culture solution was collected and the cell pellets were resuspended in SDS–PAGE sample loading buffer. A total of 280 ml cultures was centrifuged at the speed of 5000 rpm for 20 min, and the supernatant was centrifuged for another 50 min at 9900 rpm. The final supernatant was collected and filtered with a 0.22 μm pore size filter (Millipore, MA). All the proteins were collected by filtrating through a nitrocellulose filter (BA85, Whatman, Germany) three times. The filter was dissolved in 100 μl SDS sample buffer for 15 min at 65 °C and then boiled for 10 min to recover the protein present. Protein samples of both total cell pellet and culture supernatant were resolved by SDS–PAGE and detected by western blot analysis. All samples were normalized to the OD₆₀₀ of the culture and volume used in the preparation. Secretion assays for YPK_0952 were carried out by a similar procedure.

Western blot analysis

Protein samples were resolved by SDS–PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, MA). Then the membrane was blocked in 5% (w/v) BSA for 8 h at 4 °C, and incubated with primary antibodies at 4 °C overnight: anti-VSVG (Santa Cruz biotechnology, catalog no. sc-365019, lot number: B0916), 1:1000; anti-ICDH⁴⁷,1:6000; anti-RNAP (Santa Cruz biotechnology, catalog no. sc-56766, lot number: F2514), 1:400; anti-His (Santa Cruz biotechnology, catalog no. sc-8036, lot number: I1018), 1:500; anti-GST (Santa Cruz biotechnology, catalog no. sc-53909, lot number: F2413), 1:500; anti-β-lactamase (Santa Cruz biotechnology, catalog no. sc-66062, lot number: 8A5.A10),1:1000. The membrane was washed five times in TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4), then incubated with 1:5,000 diluted horseradish peroxidase conjugated secondary antibodies (Shanghai Genomics, catalog no. DY60203, lot number: 20614) for 4 h at 4 °C, and washed further five times with TBST buffer. Signals were detected by using the ECL plus kit (GE Healthcare, Piscataway, NJ) with a Chemiluminescence imager (Tanon 5200Multi, Beijing).

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from exponentially growing strains using the RNAprep Pure Cell/Bacteria Kit (TIANGEN, Beijing, China) along with the DNase I Kit (Sigma-Aldrich, Taufkirchen, Germany). The concentration of RNA was measured by NanoDrop 2000 (Thermo Fisher Scientific, USA). The TransStart Green qPCR Super-Mix (TransGen Biotech, Beijing, China) and the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, USA) was used to measure mRNA abundance in each of the samples according to manufacturer’s instructions. Primers used in this study are list in Supplementary Table 2. To normalize the results, the relative abundance of 16S rRNA was used as an internal standard.

DNase assay

Purified Tce1 protein (0.016 μM) was incubated with λ DNA (0.35 μg, Takara, Japan, catalog no. 3010) in the reaction buffer (20 mM MES, 100 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, pH 6.9). In all, 4 mM EDTA, 2 mM other divalent metal or other component was added in the reaction system as indicated in different experiments. The reaction of DNA hydrolysis was carried out at 37 °C for 30 min or indicated time points and the integrity of DNA was analyzed by 0.7% agarose gel electrophoresis.

RNase assay

Total RNA was extracted from E. coli TG1 and tRNA from E. coli MRE 600 (Roche, Germany, catalog no. 10109541001) was purchased from Sigma-Aldrich. Two micrograms of RNA was incubated with different concentrations of Tce1 in same reaction system as DNase assay at 37 °C for 30 min. The integrity of RNA was detected by 2% agarose gel.

Fluorophore labeling of proteins

Fluorophore labeling of proteins was performed as described²⁸ with minor modifications. Cysteine residues were present in the C-terminus of both Tce1 and Tse1. To prepare the proteins for subsequent labeling reactions, 5 mM DTT was used to reduce the potential disulfide bonds formed by these cysteine residues, and the reactions were conducted at room temperature for 2 h. After the reduction of disulfide bonds, DTT is removed by dialysis in 20 mM potassium phosphate (pH 7.0) and 500 mM NaCl. Labeling reactions were carried out by adding 10 mM maleimide fluorophores (Thermo Fisher Scientific, USA, catalog no. A10254) dissolved in dimethyl sulfoxide into reduced protein at a molar ratio of 5:1 (maleimide: protein), followed by 4 °C incubation in the dark overnight. The reaction was quenched by adding 2 mM DTT and dialyzed into 2 L of 20 mM potassium phosphate (pH 7.0) and 500 mM NaCl overnight at 4 °C.

Fluorescent labeling of live bacteria

Fluorescent labeling of live bacterial strains was performed according to described methods²⁸ with some modifications. Briefly, cultures at OD₆₀₀ = 0.7 were centrifuged and resuspended in M9-glucose containing 1 μM fluorophore-conjugated protein, incubated in the dark at room temperature for 30 min. The cells were washed five times to remove the free label and resuspended in 100 μl volume in M9-glucose. Ten microliters of the cell suspension was dispensed onto 1% (w/v) agarose pads on a microscope slide before sealing with a clean glass coverslip. The result was obtained by high-speed rotary disc type fluorescence confocal microscope (Andor Revolution-XD, UK).

TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and flow cytometry analysis

Overnight culture of E. coli BL21(DE3) containing the pET28a plasmid or its derivatives expressing Tce1 alone (pET28a-tce1) or Tce1-Tci1 together (pET28a-tce1-tci1) were diluted 100-fold into LB broth and incubated at 26 °C with 180 rpm shaking. After incubated at 26 °C for 2 h, the expression of toxin and immunity genes was induced by addition of 0.5 mM IPTG and continue cultivation for 4 h at 26 °C. Collected cells were washed with PBS, fixed, incubated for 5 min in PBS with 0.3% Triton X-100 and stained using One-step TUNEL cell apoptosis detection kit (Beyotime Biotechnology, China). When genomic DNA breaks, exposed 3’-OH can be labeled with green fluorescent probe FITC catalyzed by terminal deoxynucleotidyl transferase (TdT), which can be detected by flow cytometry (Beckman, CytoFLEX). Ten-thousand cells were gathered for each sample and analyzed by FlowJo_V10⁴⁸.

DAPI staining and flow cytometry analysis

To perform DAPI staining and flow cytometry analysis⁴⁹, overnight culture of E. coli BL21(DE3) containing the pET28a plasmid or its derivatives expressing Tce1 alone (pET28a-tce1) or Tce1-Tci1 together (pET28a-tce1-tci1) were diluted 100-fold into LB broth and incubated at 26 °C with 180 rpm shaking. After incubated at 26 °C for 2 h, the expression of toxin and immunity genes was induced by addition of 0.5 mM IPTG and continue cultivation for 4 h at 26 °C. Collected cells were washed with PBS, fixed, incubated for 5 min in PBS with 0.3% Triton X-100 stained using 10 μg ml⁻¹ DAPI for 30 min at 37 °C (Solarbio, China), then washed three times with PBS and detected by fluorescence microscope (Andor Revolution-XD, Britain) or flow cytometry (Beckman, CytoFLEX). Twenty-thousand cells were gathered for each sample and analyzed by FlowJo_V10.

Subcellular fractionation

To perform subcellular fractionation⁵⁰, 2 ml overnight grown Yptb culture (OD₆₀₀ 1.0) was collected, washed, and incubated in 2 ml M9 containing 0.05 mg Tce1 at 30 °C for 60 min. Tce1-treated bacterial cells were washed with PBS to remove extracellular Tce1 protein, and incubated into 285 µl sucrose buffer (20 mM PBS, pH 7.4, 20% sucrose, 2.5 mM EDTA) for 20 min at room temperature. After that, 285 µl ice-cold 0.5 mM MgCl₂ was added and incubated for 5 min with gentle agitation. The suspension was centrifuged at 7000 × g for 20 min at 4 °C to collect the supernatant containing periplasmic proteins (Peri). The pellet was resuspended in SDS-loading buffer and defined as cytoplasmic (Cyto). All the samples were examined by SDS–PAGE and western blotting analysis.

Construction of mutant library by epPCR

Error-prone PCR (epPCR) was conducted on plasmid pET28a-tce1 by using the QuickMutation™ Random Mutagenesis Kit (Beyotime Biotechnology, China) with primers tce1-F-BamHI and tce1-R-SalI according to manufacturer’s instructions. The epPCR program was as follows: 94 °C for 3 min, 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by 10 min at 72 °C final extension. The PCR products were gel-purified, digested with BamHI and SalI, and cloned into similarly digested pET28a. The ligation mixture was transformed into BL21(DE3). Transformants lost toxicity were screened in LB medium containing 0.5 mM IPTG and were further verified by cloning the mutated alleles of tce1 into new vector⁵¹. The mutations were identified by DNA sequencing analysis.

Intra-species and inter-species competition in vitro

For intra-species competition assays²⁴, overnight grown strains were washed and adjusted to OD₆₀₀ of 1.0 with M9 medium before mixing for competition. The initial donor-to-recipient ratio was 1:1 and the co-cultures were either spotted onto a 0.22 μm nitrocellulose membrane (Nalgene) placed on M9 agar plates at 26 °C for 48 h (for contact-dependent competition), or inoculated into 2 ml M9 medium at 26 °C with shaking for 24 h or 48 h (for contact-independent competition in liquid medium). For contact-independent competition performed on a solid surface, 5 μl of the recipient strain was spotted on 0.22 μm nitrocellulose membrane on M9 agar plates. After the bacterial solution was dried, another 0.22 μm nitrocellulose membrane was put on it and 5 μl of the donor strain was spotted on the same place of the second membrane and incubated at 26 °C for 48 h. The donor and recipient strains were labeled with pKT100 (Km^R) or pACYC184 (Cm^R), respectively, to facilitate screening on YLB plates. At indicated time points after the competition, the CFU ratio of the donor and recipient strains was measured by plate counts. Data from all competitions were analyzed using the Student’s t-test, and the results shown represent the mean of one representative assay performed in triplicate.

For inter-species competition assays, overnight grown Yptb strains harboring pKT100 (Km^R) and E. coli (DH5α) or S. Typhimurium strains containing pBBRMCS5-GFP (Gm^R) or pME6032 (Tet^R) (gentamycin or tetracycline resistance) were washed three times with M9 medium, and adjusted to OD₆₀₀ = 1.0. Yptb strains diluted to 10-folds and target strains attenuated to 100-folds were mixed together so that the ratio of donor and recipient was 10 to 1 in M9 liquid, incubated at 26 °C with the speed of 120 rpm. After the competition, mixtures were serially diluted, counted on LB plates containing appropriate antibiotics, and the final CFU was determined.

Murine infection and in vivo competition assays

Female 6-week-old BALB/c mice were adapted in the lab for 3 days and orally gavaged with 10⁹ CFUs of the indicated Yptb strains labeled by pKT100 (Km^R) and monitored for 24 or 48 h. When indicated, mice were orally gavaged with streptomycin (100 μl of 200 mg ml⁻¹ solution) 24 h prior to Yptb infection. At the end of the experiment animals were sacrificed, and the cecum and small intestine tissue were ground, plated on selective YLB antibiotic plates for CFU enumeration.

For competition assays between Yptb and E. coli in mouse gut, female 6-week-old BALB/c mice were orally gavaged with streptomycin (100 μl of 200 mg ml⁻¹ solution) on day 1. On day 2, 5 × 10⁸ CFU E. coli DH5α containing GFP was gavaged, and on day 3, 5 × 10⁸ CFU of Yptb strains was orally gavaged. After 24 h on day 4 and 48 h on day 5, mice were sacrificed and cecum and small intestine tissue were separated, serial diluted, spread on YLB (nalidixic acid, for selection of Yptb) or LB (gentamicin, for selection of E. coli) plates for CFU enumeration.

For competition assays between Yptb and S. Typhimurium, due to the difficulty of detecting S. Typhimurium in cecum tissues after 12 h, mice pre-treated with streptomycin for 2 days were orally gavaged with a mixture of equal bacterial count (5 × 10⁸ CFU) of Yptb and S. Typhimurium containing pME6032. Eight hours later, mice were sacrificed, cecum and small intestine tissue were separated, serial diluted, and spread on YLB (nalidixic acid, for selection of Yptb) or XLT4⁵² (tetracycline, for selection of S. Typhimurium) plates for CFU enumeration.

Statistics and reproducibility

Statistical analyses were performed using GraphPad Prism Software (GraphPad Prism 7.00). All experiments were performed in at least three independent replicates. Statistical analyses of colonization assay in mice, intra-species and inter-species competition assay in mice were analyzed using two-sided Mann–Whitney test. All other experiments were analyzed using unpaired, two-tailed Student’s t-test. Statistical significance is determined when P < 0.05. All gels, blots, and micrographs were repeated for at least three times independently with similar results.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.