Journal: Cell reports
Article Title: A prophage-expressed type IV pilus component provides anti-phage defense
doi: 10.1016/j.celrep.2025.116759
Figure Lengend Snippet: (A) BacTH assays were performed by co-expressing JBD68 P-FimU or Pae strain PAO1 FimU (T25 fusion) with the minor pilin proteins, PilA, and T4P motor proteins PilU and PilB (T18 fusion). Interactions were detected by spotting cells on X-Gal and MacConkey maltose agar. (B) Percentage of cells making T4P in indicated strains as detected by fluorescence microscopy is shown. Each data point represents a biological replicate, and the bar graphs indicate the mean ± SD. Statistics were determined by Tukey’s multiple comparisons test. (C) Percentage of cells with polar immunofluore-scence signal in the indicated strains is shown. Each data point represents a biological replicate, and the bar graphs indicate the mean ± SD. Statistics were determined using Student’s t test. ns, not significant. (D) Fluorescence microscopy images of cells from populations of indicated strains are shown with immunolabeled FimU and P-FimU (magenta) associated with T4P (green) labeled with AF488-maleimide. Arrows indicate pole- and pilus-associated FimU. Scale bars, 2 μm.
Article Snippet: Difco MacConkey Agar Base , BD , Cat# 281810.
Techniques: Expressing, Fluorescence, Microscopy, Immunolabeling, Labeling
Figure S1 G; n is between 731 and 1,120 cells). Statistical analysis for the differences between WT and the mutants was conducted using the unpaired t test based on 12 fields. ∗ p < 0.01, ∗∗ p < 0.001, and ∗∗∗ p < 10 −9 . (E) Close-up on the β turn in EI, right after the linker, in the 3D structure model (see C). EIN is colored in orange, the linker between the domains in purple, and EIC in green. G266 is depicted as a red sphere. (F) Images of cells expressing TmaR-YFP (yellow) and mCherry-tagged WT EI or G266C (red), all expressed from their respective native promoter and locus in the chromosome. Scale bars, 2 μM (see the respective results with EI variants G266S and G226D in
Figure S3 A). (G) Fraction of cells in the images shown in (F) and Figure S3A with mCherry-tagged EI variants (red bars) and TmaR-YFP (yellow bars) calculated based on fluorescence microscopy. The bars show the SDs ( n is between 467 and 1,175 cells). Statistical analysis for the differences between WT and the mutants was conducted for each protein using the t test based on three fields. ∗ p < 0.001. (H) Cells expressing mCherry-tagged EI, WT, or G266C, from the native pts promoter and locus in the chromosome, and harboring a plasmid expressing TmaR from an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible promoter, grown with increasing concentrations of IPTG (0, 0.1, or 1 mM) were imaged. The CVIs of the respective EI variants were calculated based on fluorescence intensity throughout each cell. Violin plots depict the CVI distribution for mCherry-tagged EI variants, with the blue line showing the median value. Between 360 and 850 cells were analyzed per variant. Representative cells above each violin illustrate the quantitative data. Scale bars, 2 μm. The same analysis for EI variants G266S and G226D in provided in
Figure S3 B. (I) CoIP of the EI variants with TmaR. Cells deleted for tmaR and expressing mCherry-tagged WT EI or one of the three EI-G266 mutant proteins from the native pts promoter and locus in the chromosome, and harboring a plasmid expressing TmaR-His or just the His tag, were crosslinked by formaldehyde. TmaR or the His tag were immunoprecipitated using nickel beads. Samples were fractionated by SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with anti-mCherry antibody to detect EI-mCherry, followed by stripping of the membrane and probing it with anti-His antibody to detect TmaR-His. The lower panel shows TmaR-His and the mCherry-tagged EI variants that were co-precipitated with it, detected by anti-His and anti-mCherry antibodies, respectively, on a representative membrane. The upper panel shows the ratio between the band intensities of mCherry-tagged EI mutants expressed in the presence of TmaR-His minus the intensities in the absence of TmaR-His and the intensity of TmaR-His band. The signal obtained when WT TmaR and WT EI are co-expressed is defined as 1. The bars show the SDs between three biological replicates. Statistical analysis for the differences between WT and the mutants was conducted using the two-sided Mann-Whitney test. ∗ p < 0.05. " width="100%" height="100%">
Journal: Cell Reports
Article Title: Uncovering the mechanism for polar sequestration of the major bacterial sugar regulator by high-throughput screens and 3D interaction modeling
doi: 10.1016/j.celrep.2025.115436
Figure Lengend Snippet: Isolation of EI mutants that are functional but do not cluster (A) The screen scheme. A library of EI mutants was constructed by error-prone PCR of the ptsI gene, fused to mCherry, present on a low-copy plasmid in the context of the entire pts operon with its native promoter. The library was introduced into MG1655 cells deleted for the pts operon. Out of 3,552 colonies that were streaked on MacConkey-glucose plates, 2,304 generated red color, indicating that they express functional EI, which enables the cells to consume the sugar. These mutants were screened by automated fluorescence microscopy to identify those that express EI protein that does not cluster at the poles. The six mutants expressing functional and diffuse EI were sequenced. (B) The screen results. The position of each amino acid that has been replaced in the six functional and mislocalized EI variants is shown. Each row shows the mutated residue(s)in one of the mutants, represented by dots, along the x axis. An orange background marks EIN, purple background marks its linker region, and green background marks EIC. (C) The 3D structure of the EI dimer (PDB: 2HWG). The two models on the left are cartoons showing the EI 3D structure from different angles, and the one on the right shows the protein surface. EIN is colored in orange, the linker region in purple, and EIC in green. Amino acids whose substitution yielded functional but mislocalized EI mutants are marked in red. G266 in both subunits is presented as a red sphere. (D) Fraction of cells with clusters of mCherry-tagged WT EI or EI mutants with single point mutations, all expressed from the pts native promoter and locus in the chromosome. The bars show the standard deviations (see representative images for all EI variants in Figure S1 G; n is between 731 and 1,120 cells). Statistical analysis for the differences between WT and the mutants was conducted using the unpaired t test based on 12 fields. ∗ p < 0.01, ∗∗ p < 0.001, and ∗∗∗ p < 10 −9 . (E) Close-up on the β turn in EI, right after the linker, in the 3D structure model (see C). EIN is colored in orange, the linker between the domains in purple, and EIC in green. G266 is depicted as a red sphere. (F) Images of cells expressing TmaR-YFP (yellow) and mCherry-tagged WT EI or G266C (red), all expressed from their respective native promoter and locus in the chromosome. Scale bars, 2 μM (see the respective results with EI variants G266S and G226D in Figure S3 A). (G) Fraction of cells in the images shown in (F) and Figure S3A with mCherry-tagged EI variants (red bars) and TmaR-YFP (yellow bars) calculated based on fluorescence microscopy. The bars show the SDs ( n is between 467 and 1,175 cells). Statistical analysis for the differences between WT and the mutants was conducted for each protein using the t test based on three fields. ∗ p < 0.001. (H) Cells expressing mCherry-tagged EI, WT, or G266C, from the native pts promoter and locus in the chromosome, and harboring a plasmid expressing TmaR from an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible promoter, grown with increasing concentrations of IPTG (0, 0.1, or 1 mM) were imaged. The CVIs of the respective EI variants were calculated based on fluorescence intensity throughout each cell. Violin plots depict the CVI distribution for mCherry-tagged EI variants, with the blue line showing the median value. Between 360 and 850 cells were analyzed per variant. Representative cells above each violin illustrate the quantitative data. Scale bars, 2 μm. The same analysis for EI variants G266S and G226D in provided in Figure S3 B. (I) CoIP of the EI variants with TmaR. Cells deleted for tmaR and expressing mCherry-tagged WT EI or one of the three EI-G266 mutant proteins from the native pts promoter and locus in the chromosome, and harboring a plasmid expressing TmaR-His or just the His tag, were crosslinked by formaldehyde. TmaR or the His tag were immunoprecipitated using nickel beads. Samples were fractionated by SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with anti-mCherry antibody to detect EI-mCherry, followed by stripping of the membrane and probing it with anti-His antibody to detect TmaR-His. The lower panel shows TmaR-His and the mCherry-tagged EI variants that were co-precipitated with it, detected by anti-His and anti-mCherry antibodies, respectively, on a representative membrane. The upper panel shows the ratio between the band intensities of mCherry-tagged EI mutants expressed in the presence of TmaR-His minus the intensities in the absence of TmaR-His and the intensity of TmaR-His band. The signal obtained when WT TmaR and WT EI are co-expressed is defined as 1. The bars show the SDs between three biological replicates. Statistical analysis for the differences between WT and the mutants was conducted using the two-sided Mann-Whitney test. ∗ p < 0.05.
Article Snippet: MacConkey plates were made by mixing 40 mg/mL of Difco MacConkey Agar base (Becton Dickinson and company) with 0.4% fructose or glucose as indicated.
Techniques: Isolation, Functional Assay, Construct, Plasmid Preparation, Generated, Fluorescence, Microscopy, Expressing, Residue, Variant Assay, Mutagenesis, Immunoprecipitation, SDS Page, Membrane, Stripping Membranes, MANN-WHITNEY
Journal: Cell Reports
Article Title: Uncovering the mechanism for polar sequestration of the major bacterial sugar regulator by high-throughput screens and 3D interaction modeling
doi: 10.1016/j.celrep.2025.115436
Figure Lengend Snippet: The screen-isolated mutations in TmaR and EI impair their interaction and the effect of isolated TmaR mutants on EI function (A) CoIP of the TmaR variants with EI. Cells deleted for the tmaR gene and expressing EI-mCherry from the pts native promoter and locus in the chromosome, as well as one of the His-tagged TmaR variants or just the His tag from a plasmid, were crosslinked by formaldehyde. The His-tagged proteins were precipitated with nickel beads and fractionated by SDS-PAGE, and the amount of EI-mCherry that co-precipitated with them was monitored by western blot analysis using anti-mCherry antibodies. The membrane was then stripped and probed with anti-His antibody to detect the TmaR-His variants. The lower panel shows the levels of EI-mCherry and the His-tagged TmaR variants that were co-precipitated with it on a representative membrane. The upper panel shows the ratio between the band intensity of EI-mCherry expressed in the presence of or TmaR-His minus the band intensity of EI-mCherry expressed in the absence of TmaR-His and the intensities of His-tagged TmaR mutant bands. The ratio obtained when WT TmaR and WT EI are co-expressed is defined as 1. The bars show the SDs between three biological replicates. Statistical analysis for the differences between WT and the mutants was conducted using the two-sided Mann-Whitney test. ∗ p < 0.05. (B) Far-western analysis of the interaction between EI and TmaR variants. Equal amounts of lysates of Δ tmaR cells overexpressing the His tag only or His-tagged WT TmaR or mutant proteins were fractionated on three SDS-polyacrylamide gels and blotted onto nitrocellulose membranes, which were probed with either anti-His antibody (western) or first with EI-mCherry or just mCherry (I) followed by anti-mCherry antibody (II). The observed bands match His-TmaR size. Of note, none of the TmaR variants interacted with the mCherry protein, which served as a negative control. The upper panel shows the ratio between the band intensity of EI-mCherry to His-tagged TmaR variant bands. The ratio obtained when WT TmaR and WT EI are co-expressed is defined as 1. The bars show the SDs between three biological replicates. Statistical analysis for the differences between WT and the mutants was conducted using the two-sided Mann-Whitney test. ∗ p < 0.05. (C) The upper panel shows images of yeast cells co-expressing variants of both mCherry-tagged EI and Venus-tagged TmaR. From left to right: EI (WT) with TmaR (WT); EI (G266C) with TmaR (WT); EI (WT) with TmaR (F8L, V11I, R16P), termed TmaR (patch-I mut); and EI(WT) with TmaR (D87H, A84G, E85D), termed TmaR (patch-II mut). Scale bars, 5 μm. The lower panel shows the phase diagrams of yeast cells expressing the indicated E. coli EI and TmaR variant proteins as functions of median fluorescence intensities of individual cells in the RFP and GFP channels. Red and black dots indicate cells with or without a TmaR condensate, respectively. (D) Examining the effect of the mutations in TmaR on EI function. A representative MacConkey plate supplemented with 0.4% fructose and containing 1 mM IPTG streaked with ΔtmaR cells harboring a plasmid that expresses WT TmaR, one of TmaR variants that cause EI mislocalization or the His tag only (EV). Red color indicates sugar consumption and, hence, that EI is functional. Cells deleted for the ptsI gene, which encodes EI (Δ ptsI ), grow as white colonies, providing a negative control. (E) The rate of glucose consumption by cells deleted for the pts operon and for the tmaR gene and expressing each of the indicated His-tagged TmaR mutants, or by cells expressing only a His tag. The bars show the CVs between four biological repeats. Statistical analysis of the rate of glucose consumption between the mutant and WT TmaR proteins was conducted using the t test. ∗∗∗ p < 10 −6 , ∗∗ p < 0.01, and ∗ p < 0.05.
Article Snippet: MacConkey plates were made by mixing 40 mg/mL of Difco MacConkey Agar base (Becton Dickinson and company) with 0.4% fructose or glucose as indicated.
Techniques: Isolation, Expressing, Plasmid Preparation, SDS Page, Western Blot, Membrane, Mutagenesis, MANN-WHITNEY, Negative Control, Variant Assay, Fluorescence, Functional Assay
