e coli strain k12  (ATCC)

Journal: PLOS Biology

Article Title: Uracil-driven ROS signaling and larval TrpA1-B neuron activation are required for priming the adult Drosophila gustatory response to bacteria

doi: 10.1371/journal.pbio.3004042

Figure Lengend Snippet: (A) Live L. brevis are necessary and sufficient to prime the larvae. Sterilized eggs were exposed to live or dead bacteria as well as supernatant. The resulting adults were raised on antibiotics-containing media (ATB). PER index of w − flies to control solutions of sucrose (Sucrose 1 mM) and sucrose + PGN from E. coli K12 (Sucrose 1mM + PGN200) were obtained. (B) Uracil is necessary and sufficient to prime the larvae. Sterilized eggs were exposed to the different treatments and the resulting adults raised on antibiotics-containing media (ATB). Specifically, eggs were either monoassociated with bacterial strains, or exposed to LB media or to LB media containing uracil or cytosine. Addition of uracil or Ecc 15 24h after sterilized egg deposition was tested. The resulting adults were raised on antibiotics-containing media (ATB). PER index of w − flies to control solutions of sucrose and sucrose + PGN from E. coli K12 at 200 µg/mL (PGN200) were obtained. (C) The Duox enzyme is necessary in enterocytes for the priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of GAL4 ( mex - GAL4 /+) and UAS (+/UAS-Duox_IR ) control flies as well as animals with the Duox transcript impaired via RNAi in enterocytes ( mex - GAL4 / UAS-Duox_IR ) were calculated. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (D) Graphical representation of the life periods during which flies are shifted from 18 °C to 29 °C for a stage-dependent RNAi assay. The ubiquitously expressed Tub-G80 ts , that inhibits the activity of GAL4, is temperature-sensitive: the GAL4 inhibitor is active at 18 °C (green) and inactivated at 29 °C (black), its inactivation allows the expression of any UAS . In the case of mex - GAL4 ; TubG80 ts /UAS-Duox_IR , at 18 °C the Duox transcripts will not be impaired (Duox) while it will be at 29 °C. (E) Duox activity in enterocytes is necessary during the larval life for priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of mex-GAL4; TubG80 ts /UAS-Duox_IR flies with the Duox transcript impaired all-life long, only during the larval stages or only during the adult stage were calculated. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (F) H 2 O 2 is detectable within the larvae 24 h following exposure to uracil 1 mM. Sterilized eggs were exposed to LB media or to LB media containing uracil. Twenty-four h later, the concentration of H 2 O 2 within the animals was quantified. To confirm that H 2 O 2 is the quantified product, catalase (0.01 U/mL and 0.001 U/mL) was applied to the samples from uracil-exposed larvae or overexpressed in enterocytes ( mex-GAL4/UAS-Catalase ). (G) Reducing the amount of ROS impairs the priming. PER index to solutions of sucrose and sucrose + PGN (PGN200) of UAS (+/UAS-Catalase ) control flies as well as animals with the Catalase over-expressed in enterocytes ( mex-GAL4/UAS-Catalase ) were obtained. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. (H) H 2 O 2 supplementation to larvae is not sufficient to prime while vitamin C (VitC) addition during the larval life prevents it. PER index to solutions of sucrose + PGN (PGN200) of w − flies was calculated. Larvae were raised on conventional media with or without vitamin C (0.2 mg/mL) or on monoassociation media with or without H 2 O 2 (1% on eggs or 1% on eggs then 1% on 24h-larvae (2X) or 2% on eggs). All the resulting adults were raised on antibiotics-containing media. PER index of w − flies to solutions of sucrose + PGN from E. coli K12 at 200 µg/mL (PGN200) was obtained. The PER index is calculated as the percentage of flies tested that responded with a PER to the stimulation ± 95% confidence interval (CI). A PER value of 1 means that 100% of the tested flies extended their proboscis following contact with the mixture, a value of 0.2 means that 20% of the animals extended their proboscis. The number of tested flies ( n ) is indicated on top of each bar. For each condition, at least 3 groups with a minimum of 10 flies per group were used. Each independent group is represented as an open circle. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001 Fisher’s exact test. Further details can be found in the detailed lines, conditions and, statistics for the figure section. The data underlying this Figure can be found in . Created in BioRender. Kurz, L. (2026). https://core.local.biorender.dev/api/short-link/9ojribp .

Article Snippet: PGN is E. coli K12: Invivogen, catalog code #tlrl-kipgn.

Techniques: Bacteria, Control, RNAi Assay, Activity Assay, Inactivation, Expressing, Concentration Assay

Journal: Nucleic Acids Research

Article Title: SSB-mediated enhancement of argonaute activity triggers SOS filamentation in bacteria

doi: 10.1093/nar/gkag175

Figure Lengend Snippet: Filamentation Induced by Ago Overexpression. (A, B) Morphological analysis and cell length quantification of T. thermophilus HB27 wild-type (WT), ago deletion strain (Δ ago ), Ago-overexpressing strain (HB27/pMK-slp-AGO-sGFP_OE), and vector control strain (HB27/pMK-slp-sGFP_OE) at different growth stages (OD 600 = 0.4, 0.6, and 1.2). (C, D) Morphology and cell length measurements of E. coli expressing heterologous CbAgo, TtAgo, or PfAgo. (E) Schematic representation of TtAgo truncation constructs and the corresponding filamentation phenotypes. (F) Cell length analysis of E. coli expressing full-length TtAgo or its truncated variants. The scale bars in (A, C) are 20 μm. The dashed line in (B, D, and F) means the mean value of cell length. Error bars indicate the standard deviations (SD). The numerical values in panel D represent the cell lengths of the corresponding groups, with data presented as mean ± SD. Different letters above bars indicate significant differences ( P < 0.05) according to Duncan’s test. The number of cells counted, denoted as “n”, is indicated below each panel.

Article Snippet: Protein identification and quantification were performed by searching against the UniProt databases of E. coli K12 (Taxon ID: 83 333, 6461 entries) and T. thermophilus HB27 (Taxon ID: 262 724, 2 201 entries).

Techniques: Over Expression, Plasmid Preparation, Control, Expressing, Construct

Journal: Nucleic Acids Research

Article Title: SSB-mediated enhancement of argonaute activity triggers SOS filamentation in bacteria

doi: 10.1093/nar/gkag175

Figure Lengend Snippet: Effects of Full-length TtAgo and Truncated TtAgo Variants on Septum Formation and Nucleoid Segregation in E. coli . (A) Scanning electron microscopy (SEM) analysis of non-filamentous and filamentous E. coli strains. Scale bar, 2 μm. Insets show magnified views of the regions outlined by the boxes; scale bar, 0.3 μm. The arrows indicate septum formation sites and breakage points along the filaments. (B) Nucleoid segregation pattern in E. coli strains overexpressing full-length or truncated TtAgo proteins. Representative images display differential interference contrast (DIC), DNA staining, sGFP fluorescence, and merged overlay images. Scale bar, 5 μm. The arrows in the "sGFP" column indicate sites of protein condensates, and arrows in the "DAPI" column indicate sites of nucleoid disruption or junctions.

Article Snippet: Protein identification and quantification were performed by searching against the UniProt databases of E. coli K12 (Taxon ID: 83 333, 6461 entries) and T. thermophilus HB27 (Taxon ID: 262 724, 2 201 entries).

Techniques: Electron Microscopy, Staining, Fluorescence, Disruption

Journal: Nucleic Acids Research

Article Title: SSB-mediated enhancement of argonaute activity triggers SOS filamentation in bacteria

doi: 10.1093/nar/gkag175

Figure Lengend Snippet: Quantitative RT-PCR analysis of SOS-response genes and rpoB in E. coli strains overexpressing full-length or truncated TtAgo proteins. ( A–D ) Expression levels of the rpoB gene and SOS response-associated genes ( recA, sulA , and lexA ) were measured by reverse transcription quantitative PCR (RT-qPCR) in E. coli strains expressing full-length or truncated forms of TtAgo. The data show means of four biological replicates. Error bars indicate the standard deviations (SD). Different letters above bars indicate significant differences ( P < 0.05) according to Duncan’s test.

Article Snippet: Protein identification and quantification were performed by searching against the UniProt databases of E. coli K12 (Taxon ID: 83 333, 6461 entries) and T. thermophilus HB27 (Taxon ID: 262 724, 2 201 entries).

Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: SSB-mediated enhancement of argonaute activity triggers SOS filamentation in bacteria

doi: 10.1093/nar/gkag175

Figure Lengend Snippet: EcSSB co-immunoprecipitates with TtAgo and enhances its activity. (A) Cleavage activity of TtAgo and its truncated mutants (ΔPIWI, ΔMP, ΔLMP) at 16°C and 37°C for 6 h. The reaction was performed using a molar ratio of protein:guide:target = 5:2:1. CK1 and CK2 represent no-protein negative controls. CK3 (positive control) refers to full-length TtAgo (1 μM) cleavage at 65°C for 1 h. CK4 is a no-guide control to assess potential non-specific cleavage by TtAgo at 37°C for 6 h. (B) DNA binding activity of TtAgo and its truncated mutants at 37°C for 30 min. Open arrowheads indicate free, unbound DNA; filled arrowheads mark the shifted bands corresponding to protein-nucleic acid complexes. The protein and nucleic acid concentrations are 0.8 and 0.2 μM, respectively. (C) Venn diagram illustrating the overlap of Ago-associated proteins identified in E. coli . The right panel shows the total proteins identified per group. (D) Cleavage activity of TtAgo at different concentrations at 37°C for 6 h, and the effect of increasing concentrations of EcSSB on TtAgo (1μM)-mediated cleavage. Numbers indicate protein concentrations. (E) Time-course analysis of TtAgo cleavage activity at 37°C following addition of EcSSB (1.4 μM) at a molar ratio of 1:1.4 (TtAgo:EcSSB). C1: no-protein negative controls. C2 (positive control): TtAgo (1 μM) cleavage at 65°C for 1 h, indicating the positions of cleavage products.

Article Snippet: Protein identification and quantification were performed by searching against the UniProt databases of E. coli K12 (Taxon ID: 83 333, 6461 entries) and T. thermophilus HB27 (Taxon ID: 262 724, 2 201 entries).

Techniques: Activity Assay, Positive Control, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: SSB-mediated enhancement of argonaute activity triggers SOS filamentation in bacteria

doi: 10.1093/nar/gkag175

Figure Lengend Snippet: The homologous recombination protein TtRad52 alleviates TtAgo-induced filamentation. (A and B) Morphology and quantification of cell length in E. coli strains expressing TtAgo alone or co-expressing TtAgo and TtRad52. The dashed lines indicate the mean cell length, and numerical values in panel B represent the cell lengths of the corresponding groups, with data presented as mean ± SD. The number of cells counted, denoted as “n,” is indicated below each panel. Statistical significance between groups was assessed using an unpaired t-test with Bonferroni correction; *** P < 0.001. Scale bar, 20 μm. (C) Fluorescently tagged TtAgo and TtRad52 show in vivo localization patterns. Scale bar, 10 μm.

Article Snippet: Protein identification and quantification were performed by searching against the UniProt databases of E. coli K12 (Taxon ID: 83 333, 6461 entries) and T. thermophilus HB27 (Taxon ID: 262 724, 2 201 entries).

Techniques: Homologous Recombination, Expressing, In Vivo