Journal: Molecular Microbiology

Article Title: Naturally Occurring CodY Variants Alter Ligand Binding, DNA Target Affinity, and Virulence in Clostridioides difficile

doi: 10.1111/mmi.70081

Figure Lengend Snippet: (A) Effect of codY alleles on toxin production in C. difficile . Toxin production (measured using ELISA) in codY mutant (UK1 codY::ErmB ) and C. difficile strains producing CodY (WT) or CodY (Y146N) or CodY (V58A). CodY production in these strains was confirmed by western blot with anti‐CodY antibodies. The membrane was stained with Ponceau stain to show equal loading of proteins. (B) Measurement of intracellular levels of c‐di‐GMP in C. difficile cytosol using the c‐di‐GMP Assay kit (Lucerna). Concentrations were calculated using a standard curve constructed with known amounts of c‐di‐GMP and the data presented indicate the c‐di‐GMP concentration in the volume (20 μL) of cytosol tested. Data represent mean ± SEM from three independent experiments. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple‐comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The cytosolic contents collected were used to measure intracellular c‐di‐GMP level using Lucerna Cyclic‐di‐GMP Assay Kit following the manufacturer's recommended protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Mutagenesis, Western Blot, Membrane, Staining, Construct, Concentration Assay, Comparison

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 for 6 h prior to gene expression analysis. Graphs present mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out

Journal: Applied and Environmental Microbiology

Article Title: Thermoregulation of functional amyloid Fap-dependent biofilm formation via cyclic diguanosine monophosphate signaling in Pseudomonas fluorescens

doi: 10.1128/aem.02387-25

Figure Lengend Snippet: fapA , fliC , and aprA expression and intracellular c-di-GMP levels are thermoregulated in P. fluorescens PF07. ( A–C ) Time course of gene expression in PF07 macrocolonies at different temperatures, as determined by qRT-PCR. The expression levels are relative to those at 28°C for 3 days. One-way ANOVA with Dunnett’s T3 multiple comparison test was used to determine statistical significance compared to the levels at 28°C for 3 days. ( D ) Relative expression levels of genes in PF07 macrocolonies after a temperature shift. PF07 colonies were cultured on tryptone plates at 15°C or 4°C for 3 days, followed by a shift to 28°C for 1 day. The expression levels determined by qRT-PCR are relative to those of control cultures maintained without a temperature shift. A two-tailed unpaired t -test with Welch’s correction was used to compare expression levels between the samples with and without a temperature shift. The gene expression data are mean ± SD of three biological replicates, each with three technical replicates. ( E ) Time-course quantification of intracellular c-di-GMP levels in PF07 macrocolonies cultured at different temperatures. The c-di-GMP levels are expressed as a percentage relative to those at 28°C for 3 days. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance relative to the 28°C group at each time point. ( F ) Intracellular c-di-GMP quantification in PF07 macrocolonies cultured at 15°C or 4°C for 3 days, followed by a shift to 28°C for 1 day. The c-di-GMP levels are expressed as a percentage relative to those of control cultures maintained without a temperature shift. A two-tailed unpaired t -test was used to determine statistical significance. In panels E and F, the intracellular c-di-GMP levels were determined using a cyclic-di-GMP assay kit (Lucerna, USA). Data are representative of two independent experiments with three technical replicates each and are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: The dried samples were dissolved in RNase-free water to analyze the c-di-GMP content using a c-di-GMP assay kit (Lucerna, USA).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Comparison, Cell Culture, Control, Two Tailed Test

Journal: Applied and Environmental Microbiology

Article Title: Thermoregulation of functional amyloid Fap-dependent biofilm formation via cyclic diguanosine monophosphate signaling in Pseudomonas fluorescens

doi: 10.1128/aem.02387-25

Figure Lengend Snippet: Low temperature represses ECM production, fapA transcription, and intracellular c-di-GMP content in macrocolony biofilms of P. fluorescens PF07 grown on sterile salmon muscle juice agar plates. ( A ) Quantification of ECM content in PF07 macrocolony biofilms cultured at different temperatures via the Congo red binding assay. ECM levels are percentages relative to those measured at 28°C for 3 days. Data are presented as mean ± SD of three biological replicates with three technical replicates each. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance compared to the 28°C group at each time point. ( B ) qRT-PCR analysis of fapA transcription levels in 3-day-old macrocolony biofilms of PF07 cultured at different temperatures. The expression levels are relative to those at 28°C. Data are mean ± SD of three biological replicates with three technical replicates each. One-way ANOVA with Dunnett’s T3 multiple comparison test was used to determine statistical significance compared to the levels at 28°C. ( C ) Quantification of intracellular c-di-GMP levels in PF07 macrocolonies cultured at different temperatures. The c-di-GMP levels are expressed as a percentage relative to those at 28°C for 3 days. Data are mean ± SD of two biological replicates with three technical replicates each. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance compared to the 28°C group at each time point. ** P < 0.01, **** P < 0.0001.

Article Snippet: The dried samples were dissolved in RNase-free water to analyze the c-di-GMP content using a c-di-GMP assay kit (Lucerna, USA).

Techniques: Sterility, Cell Culture, Binding Assay, Quantitative RT-PCR, Expressing, Comparison

Journal: Applied and Environmental Microbiology

Article Title: Thermoregulation of functional amyloid Fap-dependent biofilm formation via cyclic diguanosine monophosphate signaling in Pseudomonas fluorescens

doi: 10.1128/aem.02387-25

Figure Lengend Snippet: DebA and BifA are critical for regulating intracellular c-di-GMP levels, fapA expression, and biofilm formation in P. fluorescens PF07. ( A ) Domain architecture prediction of WspR, YfiA, DebA, BifA, and RbdA in PF07 (GenBank accession numbers XTS04226.1 , XTS04227.1 , XTS04228.1 , XTS04229.1 , and XTS04230.1 ) using the publicly available SMART algorithm . The REC (receiver) domain is conserved in response regulators; the HAMP domain (found in histidine kinases, adenylyl cyclases, methyl-binding proteins, and phosphatases) is a signal transduction domain; the GGDEF (diguanylate cyclase) domain is conserved in proteins that produce c-di-GMP from two molecules of GTP; the EAL domain is found in c-di-GMP-degrading phosphodiesterases; the PAS domain is a signal transduction module, and the PAC domain contributes to the PAS fold—these two domains are often combined and collectively termed the PAS domain . Predicted membrane-spanning helices are shown as blue vertical boxes. ( B ) Intracellular c-di-GMP levels in strains overexpressing wspR , yfiA , debA , bifA , or rbdA . The five genes were expressed from the IPTG-inducible vector pMMB206Gm in PF07. The strains were cultured at 28°C on tryptone agar plates with 0.5 mM IPTG for 2 days. ( C ) Intracellular c-di-GMP levels were quantified in wspR , yfiA , debA , bifA , or rbdA mutants cultured on tryptone agar plates at 28°C for 2 days. ( D ) fapA transcription levels measured using a β-galactosidase activity assay. The reporter plasmid MCS5-P fapA -lacZ was transferred to wild-type PF07 or the indicated mutant strains. Colonies formed after 2 days of growth at 28°C were used to measure β-galactosidase activity. c-di-GMP levels and β-galactosidase activity are percentages relative to those of the wild-type control (PF07+MMB or PF07). Data are representative of two independent experiments with three technical replicates each and are shown as mean ± SD. One-way ANOVA was used to determine statistical significance compared to the wild-type control, followed by Dunnett’s multiple comparison test ( * P < 0.05, ** P < 0.01, **** P < 0.0001; ns, not significant). ( E ) Macrocolony biofilms of wild-type PF07 and Δ wspR , Δ yfiN , Δ debA , Δ bifA , and Δ rbdA mutants on Congo red plates (scale bar = 1 cm). Representative images from at least three biological replicates are shown. ( F ) Macrocolony biofilms of the indicated strains formed on Congo red plates after 4 days of growth at 28°C (scale bar = 1 cm). ( G ) Pellicles of indicated strains formed at the air-liquid interface of static cultures after 3 days of growth at 28°C. Macrocolony biofilm and pellicle images are representative of three biological replicates. ( H ) SSA biofilm formation of the indicated strains. Biofilms were formed by strains in 96-well microplates cultured at 28°C for 24 and 48 h with gentle shaking (150 rpm). The biofilms were quantified by crystal violet staining (OD 595 ) and normalized to planktonic cell growth (OD 600 ). The amount of biofilm is expressed as a percentage relative to that of PF07+MCS5. Data are mean ± SD of eight biological replicates. Two-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance compared to PF07+MCS5 ( **** P < 0.0001; ns, not significant).

Article Snippet: The dried samples were dissolved in RNase-free water to analyze the c-di-GMP content using a c-di-GMP assay kit (Lucerna, USA).

Techniques: Expressing, Binding Assay, Transduction, Membrane, Plasmid Preparation, Cell Culture, Activity Assay, Mutagenesis, Control, Comparison, Gentle, Staining

Journal: Applied and Environmental Microbiology

Article Title: Thermoregulation of functional amyloid Fap-dependent biofilm formation via cyclic diguanosine monophosphate signaling in Pseudomonas fluorescens

doi: 10.1128/aem.02387-25

Figure Lengend Snippet: Temperature-dependent enzymatic activity of purified DebA, DebAΔPAS, and BifA proteins. ( A ) DGC activities of DebA and DebAΔPAS are expressed as c-di-GMP produced per nmol of purified protein per hour. ( B ) The enzymatic activities of DebA and BifA at different temperatures are presented as percentages of their activities measured at their respective optimal temperatures. Data are representative of at least two independent experiments with three technical replicates each and are shown as mean ± SD.

Article Snippet: The dried samples were dissolved in RNase-free water to analyze the c-di-GMP content using a c-di-GMP assay kit (Lucerna, USA).

Techniques: Activity Assay, Purification, Produced

Journal: Applied and Environmental Microbiology

Article Title: Thermoregulation of functional amyloid Fap-dependent biofilm formation via cyclic diguanosine monophosphate signaling in Pseudomonas fluorescens

doi: 10.1128/aem.02387-25

Figure Lengend Snippet: A temperature-dependent regulatory model for c-di-GMP-mediated biofilm formation in P. fluorescens PF07. At 28°C, high DebA expression and PAS domain-dependent catalytic activity, paired with thermolabile BifA, elevate intracellular c-di-GMP levels, which promote robust biofilm formation via the c-di-GMP/BrfA/Fap pathway. At 15°C or 4°C, reduced DebA expression and activity, alongside cold-stable BifA, decrease c-di-GMP synthesis and accelerate its degradation, leading to low intracellular c-di-GMP levels that repress fap expression and inhibit biofilm formation.

Article Snippet: The dried samples were dissolved in RNase-free water to analyze the c-di-GMP content using a c-di-GMP assay kit (Lucerna, USA).

Techniques: Expressing, Activity Assay