Journal: Molecular microbiology

Article Title: CdiA promotes receptor-independent intercellular adhesion

doi: 10.1111/mmi.13114

Figure Lengend Snippet: Bacterial strains

Article Snippet: ​ Plasmid Description a Reference pTrc99a IPTG-inducible expression plasmid, Amp R GE Healthcare pCH450 pACYC184 derivative with E. coli araBAD promoter for arabinose-inducible expression, Tet R ( Hayes & Sauer, 2003 ) pSIM6 Expresses phage λ Red proteins from heat-shock inducible promoter, Amp R ( Datta et al., 2006 ) pKAN pBluescript with FRT-flanked kanamycin-resistance cassette ligated into Smal restriction site, Amp R Kan R ( Hayes & Sauer, 2003 ) pKAN-BamA(KO) pKAN containing regions upstream and downstream of bamA Eco , Amp R Kan R This study pWEB-TNC Cosmid cloning vector, Amp R Cm R Epicentre pDsRedExpress2 Constitutive expression of DsRed, Amp R Clontech pDAL741 pBR322 derivative that expresses cdiI EC93 immunity gene, Amp R ( Aoki et al., 2005 ) pCP20 Heat-inducible expression of FLP recombinase, Cm R Amp R ( Cherepanov & Wackernagel, 1995 ) pSH21P pET21b-derived expression plasmid, appends an N-terminal His 6 epitope tag, Amp R ( Koskiniemi et al., 2014 ) pCH450-DsRed L-arabinose inducible expression of DsRed, Tet R This study pTrc99A-sfGFP IPTG inducible expression of superfolding GFP, Amp R This study pCH450-GFP L-arabinose inducible expression of superfolding GFP, Tet R This study pKAN -cdiA Δ CT Construct to delete the cdiA-CT region from E. coli EC93, Amp R Kan R This study pKAN-Δ cdiI Construct to delete the cdiI immunity gene from E. coli EC93, Amp R Kan R This study pZS21 pSC101-derived plasmid vector, Kan R ( Lutz & Bujard, 1997 ) pZS21- bamA + pZS21 derivative that expresses bamA Eco , Kan R ( Kim et al., 2007 ) pZS21- bamA ECL Expresses bamA ECL from Enterobacter cloacae ATCC 13047, Kan R ( Ruhe et al., 2013b ) PDAL660Δ1-39 Constitutive expression of the cdiBAl EC93 gene cluster, Cm R Amp R ( Aoki et al., 2005 ) pDAL878 Derivative of pDAL660Δ1-39 in which the cdiA-CT/cdiI region has been deleted.

Techniques:

Journal: Molecular microbiology

Article Title: CdiA promotes receptor-independent intercellular adhesion

doi: 10.1111/mmi.13114

Figure Lengend Snippet: Plasmids

Article Snippet: ​ Plasmid Description a Reference pTrc99a IPTG-inducible expression plasmid, Amp R GE Healthcare pCH450 pACYC184 derivative with E. coli araBAD promoter for arabinose-inducible expression, Tet R ( Hayes & Sauer, 2003 ) pSIM6 Expresses phage λ Red proteins from heat-shock inducible promoter, Amp R ( Datta et al., 2006 ) pKAN pBluescript with FRT-flanked kanamycin-resistance cassette ligated into Smal restriction site, Amp R Kan R ( Hayes & Sauer, 2003 ) pKAN-BamA(KO) pKAN containing regions upstream and downstream of bamA Eco , Amp R Kan R This study pWEB-TNC Cosmid cloning vector, Amp R Cm R Epicentre pDsRedExpress2 Constitutive expression of DsRed, Amp R Clontech pDAL741 pBR322 derivative that expresses cdiI EC93 immunity gene, Amp R ( Aoki et al., 2005 ) pCP20 Heat-inducible expression of FLP recombinase, Cm R Amp R ( Cherepanov & Wackernagel, 1995 ) pSH21P pET21b-derived expression plasmid, appends an N-terminal His 6 epitope tag, Amp R ( Koskiniemi et al., 2014 ) pCH450-DsRed L-arabinose inducible expression of DsRed, Tet R This study pTrc99A-sfGFP IPTG inducible expression of superfolding GFP, Amp R This study pCH450-GFP L-arabinose inducible expression of superfolding GFP, Tet R This study pKAN -cdiA Δ CT Construct to delete the cdiA-CT region from E. coli EC93, Amp R Kan R This study pKAN-Δ cdiI Construct to delete the cdiI immunity gene from E. coli EC93, Amp R Kan R This study pZS21 pSC101-derived plasmid vector, Kan R ( Lutz & Bujard, 1997 ) pZS21- bamA + pZS21 derivative that expresses bamA Eco , Kan R ( Kim et al., 2007 ) pZS21- bamA ECL Expresses bamA ECL from Enterobacter cloacae ATCC 13047, Kan R ( Ruhe et al., 2013b ) PDAL660Δ1-39 Constitutive expression of the cdiBAl EC93 gene cluster, Cm R Amp R ( Aoki et al., 2005 ) pDAL878 Derivative of pDAL660Δ1-39 in which the cdiA-CT/cdiI region has been deleted.

Techniques: Expressing, Plasmid Preparation, Clone Assay, Construct

Journal: Molecular microbiology

Article Title: CdiA promotes receptor-independent intercellular adhesion

doi: 10.1111/mmi.13114

Figure Lengend Snippet: A) CH9591 (bamAECL) cells were transformed with pTNC-WEB (CDI−) or pDAL660Δ1-39 (CDI+) plasmids and labeled with GFP or DsRed. Each of the four cell populations was analyzed by flow cytometry to detect background levels of dual green/red fluorescent events. B) The individual cell populations show in panel A were mixed at 1:1 ratio and incubated with shaking at 37 °C. The suspensions were then analyzed by flow cytometry using FL1 (533/30nm, GFP) and FL2 (585/40nm, DsRed) fluorophore filters.

Article Snippet: ​ Plasmid Description a Reference pTrc99a IPTG-inducible expression plasmid, Amp R GE Healthcare pCH450 pACYC184 derivative with E. coli araBAD promoter for arabinose-inducible expression, Tet R ( Hayes & Sauer, 2003 ) pSIM6 Expresses phage λ Red proteins from heat-shock inducible promoter, Amp R ( Datta et al., 2006 ) pKAN pBluescript with FRT-flanked kanamycin-resistance cassette ligated into Smal restriction site, Amp R Kan R ( Hayes & Sauer, 2003 ) pKAN-BamA(KO) pKAN containing regions upstream and downstream of bamA Eco , Amp R Kan R This study pWEB-TNC Cosmid cloning vector, Amp R Cm R Epicentre pDsRedExpress2 Constitutive expression of DsRed, Amp R Clontech pDAL741 pBR322 derivative that expresses cdiI EC93 immunity gene, Amp R ( Aoki et al., 2005 ) pCP20 Heat-inducible expression of FLP recombinase, Cm R Amp R ( Cherepanov & Wackernagel, 1995 ) pSH21P pET21b-derived expression plasmid, appends an N-terminal His 6 epitope tag, Amp R ( Koskiniemi et al., 2014 ) pCH450-DsRed L-arabinose inducible expression of DsRed, Tet R This study pTrc99A-sfGFP IPTG inducible expression of superfolding GFP, Amp R This study pCH450-GFP L-arabinose inducible expression of superfolding GFP, Tet R This study pKAN -cdiA Δ CT Construct to delete the cdiA-CT region from E. coli EC93, Amp R Kan R This study pKAN-Δ cdiI Construct to delete the cdiI immunity gene from E. coli EC93, Amp R Kan R This study pZS21 pSC101-derived plasmid vector, Kan R ( Lutz & Bujard, 1997 ) pZS21- bamA + pZS21 derivative that expresses bamA Eco , Kan R ( Kim et al., 2007 ) pZS21- bamA ECL Expresses bamA ECL from Enterobacter cloacae ATCC 13047, Kan R ( Ruhe et al., 2013b ) PDAL660Δ1-39 Constitutive expression of the cdiBAl EC93 gene cluster, Cm R Amp R ( Aoki et al., 2005 ) pDAL878 Derivative of pDAL660Δ1-39 in which the cdiA-CT/cdiI region has been deleted.

Techniques: Transformation Assay, Labeling, Flow Cytometry, Incubation

Journal: Molecular microbiology

Article Title: CdiA promotes receptor-independent intercellular adhesion

doi: 10.1111/mmi.13114

Figure Lengend Snippet: A) Strains CH9604 (bamAEco) and CH9591 (bamAECL) that carry either pTNC-WEB (CDI−) or pDAL660Δ1-39 (CDI+) were grown in LB medium and analyzed by flow cytometry for forward scatter. B) The cells from prepared for panel A were also visualized by light microscopy to determine the number of bacterial cells per aggregate.

Article Snippet: ​ Plasmid Description a Reference pTrc99a IPTG-inducible expression plasmid, Amp R GE Healthcare pCH450 pACYC184 derivative with E. coli araBAD promoter for arabinose-inducible expression, Tet R ( Hayes & Sauer, 2003 ) pSIM6 Expresses phage λ Red proteins from heat-shock inducible promoter, Amp R ( Datta et al., 2006 ) pKAN pBluescript with FRT-flanked kanamycin-resistance cassette ligated into Smal restriction site, Amp R Kan R ( Hayes & Sauer, 2003 ) pKAN-BamA(KO) pKAN containing regions upstream and downstream of bamA Eco , Amp R Kan R This study pWEB-TNC Cosmid cloning vector, Amp R Cm R Epicentre pDsRedExpress2 Constitutive expression of DsRed, Amp R Clontech pDAL741 pBR322 derivative that expresses cdiI EC93 immunity gene, Amp R ( Aoki et al., 2005 ) pCP20 Heat-inducible expression of FLP recombinase, Cm R Amp R ( Cherepanov & Wackernagel, 1995 ) pSH21P pET21b-derived expression plasmid, appends an N-terminal His 6 epitope tag, Amp R ( Koskiniemi et al., 2014 ) pCH450-DsRed L-arabinose inducible expression of DsRed, Tet R This study pTrc99A-sfGFP IPTG inducible expression of superfolding GFP, Amp R This study pCH450-GFP L-arabinose inducible expression of superfolding GFP, Tet R This study pKAN -cdiA Δ CT Construct to delete the cdiA-CT region from E. coli EC93, Amp R Kan R This study pKAN-Δ cdiI Construct to delete the cdiI immunity gene from E. coli EC93, Amp R Kan R This study pZS21 pSC101-derived plasmid vector, Kan R ( Lutz & Bujard, 1997 ) pZS21- bamA + pZS21 derivative that expresses bamA Eco , Kan R ( Kim et al., 2007 ) pZS21- bamA ECL Expresses bamA ECL from Enterobacter cloacae ATCC 13047, Kan R ( Ruhe et al., 2013b ) PDAL660Δ1-39 Constitutive expression of the cdiBAl EC93 gene cluster, Cm R Amp R ( Aoki et al., 2005 ) pDAL878 Derivative of pDAL660Δ1-39 in which the cdiA-CT/cdiI region has been deleted.

Techniques: Flow Cytometry, Light Microscopy

Journal: Nature Communications

Article Title: Tuning evolvability via plasmid copy number and regulatory architecture

doi: 10.1038/s41467-025-67995-9

Figure Lengend Snippet: Selected values of PCN correspond to the average for the pSC101 variants used in subsequent genetic constructs (i.e., 3, 5, 9, 17, 25, 39, and 79). For each value, the results of 50 independent replicates are presented. a Within each generation, first mutations are generated, then plasmids are randomly distributed after their replication. Subsequently, half of the population is selected to initialize the next generation, thus keeping the population size constant across generations. b The subpopulation harboring at least one mutated plasmid increases with PCN. For each PCN, the means from 50 independent replicates are presented across generations. Shaded areas represent 95% confidence intervals of the estimated mean values. c The number of mutated plasmids per mutant bacteria increases with PCN. d The fraction of mutated plasmids per mutant bacteria decreases with PCN.

Article Snippet: The IFFL control circuit (Addgene plasmid #109254) together with the set of pSC101 variants (Addgene plasmids #109240–#109246) are adopted from ref. . All plasmids are built using standard molecular cloning procedures , including PCR amplification, restriction digestion, gel electrophoresis, ligation, and transformation.

Techniques: Construct, Generated, Plasmid Preparation, Mutagenesis, Bacteria

Journal: Nature Communications

Article Title: Tuning evolvability via plasmid copy number and regulatory architecture

doi: 10.1038/s41467-025-67995-9

Figure Lengend Snippet: a The inducer arabinose together with AraC expressed from the genome activates EvolvR, which is harbored on a p15A plasmid to generate mutations using an engineered DNA polymerase and Cas9 nickase. The resulting fusion protein (enCas9-Poll3M-TBD) targets a 20 basepair long window at a user-defined location, specified by the sgRNA. A secondary plasmid with a variant of the pSC101 ori contains the non-fluorescent sfGFP d gene. b Following co-transformation with both plasmids, cells are induced with arabinose to activate EvolvR. Data in Supplementary Fig. highlight that the presence of arabinose or the synthesis of sfGFP d do not impact cell growth, unlike the expression of EvolvR. To mitigate this burden, we adopted 30 °C during the induction stage. We allow a flexible regrowth window for post-induction cultures that spans approximately 6 h to ensure that the cultures reach comparable growth states in exponential phase before subsequent FACS analysis for mutant detection. The mutant subpopulation is identified using flow cytometer data (Supplementary Fig. ), comparing the pre-induced and post-induced samples (Supplementary Fig. ). Created in BioRender. Gyorgy, A. (2025) https://BioRender.com/3mvgrft . c The mutant ratio is obtained in flow cytometer experiments (Supplementary Fig. ) using eight independent replicates, which is then leveraged using fluctuation analysis , to derive the phenotypic mutation rate (error bars represent 95% confidence intervals of the estimated mutation probability). See Supplementary Fig. for data on mutant ratios and pairwise statistical significance tests across PCN values. In the boxplot figure, centers indicate the median. The lower and upper bounds of the box represent the first quartile (Q1) and third quartile (Q3), respectively. With IQR = Q3–Q1 denoting the interquartile range, the whiskers extend from the box to the minimum and maximum non-outlier values such that the lower and upper thresholds are defined as Q1−1.5IQR and Q3+1.5IQR, respectively.

Article Snippet: The IFFL control circuit (Addgene plasmid #109254) together with the set of pSC101 variants (Addgene plasmids #109240–#109246) are adopted from ref. . All plasmids are built using standard molecular cloning procedures , including PCR amplification, restriction digestion, gel electrophoresis, ligation, and transformation.

Techniques: Plasmid Preparation, Variant Assay, Transformation Assay, Expressing, Mutagenesis, Flow Cytometry

Journal: Nature Communications

Article Title: Tuning evolvability via plasmid copy number and regulatory architecture

doi: 10.1038/s41467-025-67995-9

Figure Lengend Snippet: The IFFL-based control module regulates the expression of the target protein to ensure that its synthesis rate is independent of PCN. a The target gene is sfGFP d from Fig. a. b The production rate of fluorescent sfGFP per mutated plasmid decreases with PCN due to the control module, ensuring that the total production of sfGFP (including both sfGFP and sfGFP d ) remains constant across pSC101 variants. Data obtained in numerical simulations. c The average sfGFP level in mutant bacteria remains a decreasing function of PCN even after the inclusion of the control module. Data obtained in stochastic simulations, considering 50 independent replicates for each PCN. While the size of the detected mutant population increases monotonically with PCN if the detection limit is low (gray), the relationship can instead become non-monotonic as the threshold increases (green shades). Points represent mean population size and error bars indicate 95% confidence intervals. d The phenotypic mutation rate is estimated using fluctuation analysis based on the number of events above the detection limit in flow cytometer experiments when the target is sfGFP d (Supplementary Fig. ), and based on selective agar plating experiments when the target is ampR d . Mean phenotypic mutation rate is obtained from 8 independent replicates and 3 independent trials for the targets sfGFP d and ampR d , respectively. Error bars represent 95% confidence intervals. See Supplementary Figs. for data on mutant ratios and pairwise statistical significance tests across PCN values. e The target gene is ampR d .

Article Snippet: The IFFL control circuit (Addgene plasmid #109254) together with the set of pSC101 variants (Addgene plasmids #109240–#109246) are adopted from ref. . All plasmids are built using standard molecular cloning procedures , including PCR amplification, restriction digestion, gel electrophoresis, ligation, and transformation.

Techniques: Control, Expressing, Plasmid Preparation, Mutagenesis, Bacteria, Flow Cytometry

Journal: Nature Communications

Article Title: Tuning evolvability via plasmid copy number and regulatory architecture

doi: 10.1038/s41467-025-67995-9

Figure Lengend Snippet: DH5 α ΔlacI::ampR cells are co-transformed with a p15A plasmid and a pSC101 variant. Arabinose induction is reduced from 2 mM to 200 μ M to decrease the activity of EvolvR and the chance of its binding to off-target sites. Subsequent to arabinose induction, anhydrotetracycline (aTc) at a final concentration of 100 ng/mL is supplemented to relieve the effects of residual TetR. The phenotypic mutation rate is estimated using fluctuation analysis based on the number of events above the detection limit in flow cytometer experiments (Supplementary Figs. ). a To prevent leaky expression of sfGFP, a lacO array containing three LacI binding sites ( lacO x3) is placed upstream to the P tetO-lacO promoter to increase the effective local concentration of LacI – . b Expression of LacI is constitutive. Cells harboring pSC101 variants with PCN at least 25 become unstable, likely as a result of the metabolic burden due to the synthesis of LacI (Supplementary Fig. ). Points represent mean values from 8 independent replicates and error bars indicate 95% confidence intervals. See Supplementary Figs. for data on mutant ratios and pairwise statistical significance tests across PCN values. c Expression of LacI is under negative autoregulation. On-target mutations are found at a few specific positions that are functionally important, disrupting the lacO sites, resulting in the dominance of a small subset of genotypes (Supplementary Fig. ). Points represent mean values from 8 independent replicates, and error bars indicate 95% confidence intervals. See Supplementary Figs. for data on mutant ratios and pairwise statistical significance tests across PCN values (even with the inclusion of the TetR repression module and the reduced EvolvR expression, off-target mutations persist for pSC101 variants with PCN below 9, thus the corresponding data are omitted here).

Article Snippet: The IFFL control circuit (Addgene plasmid #109254) together with the set of pSC101 variants (Addgene plasmids #109240–#109246) are adopted from ref. . All plasmids are built using standard molecular cloning procedures , including PCR amplification, restriction digestion, gel electrophoresis, ligation, and transformation.

Techniques: Transformation Assay, Plasmid Preparation, Variant Assay, Activity Assay, Binding Assay, Concentration Assay, Mutagenesis, Flow Cytometry, Expressing