Journal: bioRxiv

Article Title: Uracil-Driven ROS signals activate larval TrpA1-B neurons to prime the Drosophila adult gustatory response to bacterial signal

doi: 10.64898/2025.12.22.695905

Figure Lengend Snippet: ( A ) Live L. brevis are necessary and sufficient to prime the larvae. PER index of w- flies to control solutions of sucrose and sucrose + PGN from E. coli K12 (PGN). Larvae from sterilized eggs were exposed to the different treatments and the resulting adults raised on antibiotics-containing media. ( B ) Uracil is necessary and sufficient to prime the larvae. PER index of w-flies to control solutions of sucrose and sucrose + PGN from E. coli K12 at 200µg/mL (PGN200). Larvae were monoassociated with bacterial strains, with LB media or with LB media containing uracil. The resulting adults were raised on antibiotics-containing media. (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 ). 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. Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. ( F ) 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 ). Larvae were raised on conventional media and the resulting adults raised on antibiotics-containing media. ( G ) H 2 O 2 supplementation to larvae is not sufficient to prime while vitamin C addition during the larval life prevents it. PER index to solutions of sucrose + PGN (PGN200) of w - flies. Larvae were raised on conventional media with or without vitamin C (0.2 mg/mL) or on antibiotics-containing media with or without H 2 O 2 (1%). All the resulting adults were raised on antibiotics-containing media. Labellar PER was measured to 1 mM sucrose plus or minus PGN. 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 Exact Test. Further details can be found in the detailed lines, conditions and, statistics for the figure section.

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

Techniques: Control, RNAi Assay, Activity Assay, Expressing

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

Journal: Journal of Bacteriology

Article Title: A 3D-printed capillary tube holder for high-throughput chemotaxis assays

doi: 10.1128/jb.00384-25

Figure Lengend Snippet: Constructing standard curves. Standard curves were constructed for each species in both 96- and 384-well plates by plotting the lag times of each well by the number of cells in each well. ( A ) Representative growth curves for the twofold serial dilutions of Bacillus subtilis 3610 and Escherichia coli K12 in 96- and 384-well plates. The solid lines indicate the mean of the technical replicates, while the shaded regions represent the 95% CI. Growth curves are colored by dilution with the wells with the highest concentration of bacterial cells shown in the darkest shade. For 96-well plates, three technical replicates were included for each condition. For 384-well plates, eight technical replicates were included for each condition. ( B ) Standard curves are calculated from growth curves by plotting the number of cells in each well against the lag time. Equations for the best fit line and R 2 value are shown in the figure. Results in this figure are representative of two biological replicates, each containing three or eight technical replicates, for 96- or 384-well plates, respectively.

Article Snippet: E. coli K12 (ATCC 25404) and B. subtilis 3610 (ATCC 6051) were inoculated into LB media and incubated at 37°C with shaking at 250 rpm for 16–18 hours.

Techniques: Construct, Concentration Assay

Journal: Journal of Bacteriology

Article Title: A 3D-printed capillary tube holder for high-throughput chemotaxis assays

doi: 10.1128/jb.00384-25

Figure Lengend Snippet: Ninety-six-well plate threshold concentrations. ( A ) Representative growth curve data from B. subtilis (BS) and E. coli (EC) chemotaxis experiments in 96-well plates. The solid lines indicate the mean of the six technical replicates, while the shaded regions represent the 95% CI. Growth curves are color coded by the concentration of the chemoeffector used in the sample. A legend for the color can be found below the graphs. ( B ) Calculated cell counts for B. subtilis and E. coli capillary tubes containing various concentrations of chemoeffectors. Cell counts are calculated from the growth curve data using the appropriate standard curve. Robust regression and outlier removal were used to remove outliers before plotting the data. Error bars denote a standard deviation of n = 6 technical replicates. One-way ANOVA with Dunnett’s test was used to calculate P values and compare each sample to the control. **, P < 0.01; ****, P < 0.0001; ns, P > 0.05. ( C ) Heat map showing the chemotactic index (CI) calculated for each condition. CI = T / ( T + C ), where T is the number of cells in the chemoeffector capillary, and C is the number of cells in the control capillary. CI > 0.6 is considered an attractant. Results in this figure are representative of two biological replicates, each containing six technical replicates.

Article Snippet: E. coli K12 (ATCC 25404) and B. subtilis 3610 (ATCC 6051) were inoculated into LB media and incubated at 37°C with shaking at 250 rpm for 16–18 hours.

Techniques: Chemotaxis Assay, Concentration Assay, Standard Deviation, Control

Journal: Journal of Bacteriology

Article Title: A 3D-printed capillary tube holder for high-throughput chemotaxis assays

doi: 10.1128/jb.00384-25

Figure Lengend Snippet: Three hundred eighty-four-well plate threshold concentrations. ( A ) Representative growth curve data from B. subtilis (BS) and E. coli (EC) chemotaxis experiments in 384-well plates. The solid lines indicate the mean of nine technical replicates, while the shaded regions represent the 95% CI. Growth curves are color coded by the concentration of the chemoeffector used in the sample. The legend for the color can be found below the graphs. ( B ) Cell counts are calculated from the growth curve data using the appropriate standard curve. Robust regression and outlier removal were used to remove outliers before plotting the data. Error bars denote a standard deviation of n = 9 technical replicates. One-way ANOVA with Dunnett’s test was used to calculate P values and compare each sample to the control. ** , P < 0.01; **** , P < 0.0001; ns, P > 0.05. ( C ) Heat map showing the chemotactic index (CI) calculated for each condition. CI = T / ( T + C ), where T is the number of cells in the chemoeffector capillary, and C is the number of cells in the control capillary. CI > 0.6 is considered an attractant. Boxes with white X’s are samples for which data were not collected. Results in this figure are representative of two biological replicates, each containing nine technical replicates.

Article Snippet: E. coli K12 (ATCC 25404) and B. subtilis 3610 (ATCC 6051) were inoculated into LB media and incubated at 37°C with shaking at 250 rpm for 16–18 hours.

Techniques: Chemotaxis Assay, Concentration Assay, Standard Deviation, Control

Journal: Microbiology

Article Title: Hurdles to horizontal gene transfer: species-specific effects of synonymous variation and plasmid copy number determine antibiotic resistance phenotype

doi: 10.1099/mic.0.001652

Figure Lengend Snippet: aacC1 synonymous variant design and constructs. ( a ) First 34 codons of the synonymous variant sequences. The first 10 codons (30 bp) were conserved across variants. The remaining codons were assigned different synonymous codons in a semi-random manner, guided by the codon usage of the bacterial species used. Pairwise identity with wt aacC1 is shown to the right of each sequence. ( b ) Variant constructs were composed of an integron-derived promoter and 5′ UTR, followed by the aacC1 gene and cloned into a pBBR1 broad-host-range plasmid. Variant plasmid sequences differed only by the variable region within aacC1 . ( c ) Codon usage similarity (COUSIN) for the 32 synonymous aacC1 variants in the three host species used. Variants are ordered by increasing COUSIN values in E. coli . GC content at the third base of each codon (GC3) is represented by bubble size. The five variants that could not be transformed into A. baylyi are circled in red on the x-axis. Equivalent figures ordered by increasing COUSIN values in A. baylyi and P. aeruginosa are provided as Fig. S2B and C.

Article Snippet: Synonymous variants of the aacC1 gene (GenBank: AAB20441.1 , Serratia marcescens ) were designed using the Optimization Analysis function of the COUSIN (COdon Usage Similarity INdex) tool [ ] and codon usage tables (CUTs) of E. coli K12 MG1655 (GenBank: U00096.3 ), P. aeruginosa PAO1 (ATCC 15692, NC_002516.2 ), A. baylyi ADP1 ( NC_005966.1 ) and Bacillus cereus ATCC 14579 (GenBank: AP007209.1 ).

Techniques: Variant Assay, Construct, Sequencing, Derivative Assay, Clone Assay, Plasmid Preparation, Transformation Assay

Journal: Microbiology

Article Title: Hurdles to horizontal gene transfer: species-specific effects of synonymous variation and plasmid copy number determine antibiotic resistance phenotype

doi: 10.1099/mic.0.001652

Figure Lengend Snippet: Synonymous variants confer different resistance levels. ( a ) Resistance levels are represented by IC50 AUC , i.e. the concentration of gentamicin required to reduce the AUC by 50% compared to growth in the absence of gentamicin. Synonymous variants are ordered on the x-axis by increasing IC50 AUC in E. coli . ( b ) PCN, quantified by qPCR, in each host variant. Significant differences in PCN are indicated by ***, and non-significant differences are indicated by ns. ( c ) AUC of the three bacteria species carrying the aacC1 variants in 50 µg ml −1 and 400 µg ml −1 of gentamicin. AUC is represented as relative to AUC in 0 µg ml −1 gentamicin. Some variant*species combinations were not tested at each of the concentrations. The AUC of these combinations was set to 0 when the variant*combination did not grow at a lower gentamicin concentration and to 1 when the variant*combination had, in the presence of a higher concentration of gentamicin, an AUC equivalent to the one in the absence of gentamicin. White boxes indicate the five variants that could not be transformed in A. baylyi .

Article Snippet: Synonymous variants of the aacC1 gene (GenBank: AAB20441.1 , Serratia marcescens ) were designed using the Optimization Analysis function of the COUSIN (COdon Usage Similarity INdex) tool [ ] and codon usage tables (CUTs) of E. coli K12 MG1655 (GenBank: U00096.3 ), P. aeruginosa PAO1 (ATCC 15692, NC_002516.2 ), A. baylyi ADP1 ( NC_005966.1 ) and Bacillus cereus ATCC 14579 (GenBank: AP007209.1 ).

Techniques: Concentration Assay, Variant Assay, Bacteria, Transformation Assay

Journal: Microbiology

Article Title: Hurdles to horizontal gene transfer: species-specific effects of synonymous variation and plasmid copy number determine antibiotic resistance phenotype

doi: 10.1099/mic.0.001652

Figure Lengend Snippet: CUPs and resistance. Pearson correlation between resistance (IC50 AUC ) and tAI (first line), COUSIN (second line), CAI (third line) and |Δ(GC3)| (fourth line) for A. baylyi (first column), E. coli (second column) and P. aeruginosa (third column). Significant correlations ( P <0.05) are represented by a black regression line. Non-significant Pearson correlations are represented by a grey dotted regression line.

Article Snippet: Synonymous variants of the aacC1 gene (GenBank: AAB20441.1 , Serratia marcescens ) were designed using the Optimization Analysis function of the COUSIN (COdon Usage Similarity INdex) tool [ ] and codon usage tables (CUTs) of E. coli K12 MG1655 (GenBank: U00096.3 ), P. aeruginosa PAO1 (ATCC 15692, NC_002516.2 ), A. baylyi ADP1 ( NC_005966.1 ) and Bacillus cereus ATCC 14579 (GenBank: AP007209.1 ).

Techniques: