lb liquid media  (Bacto Laboratories)

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: Mid-log phase cultures of WT (SL174; lacZ + ) and Δ oafA (SL180; lacZ - ) STm were washed in PBS, combined at a ratio of approximately 6000:1, and then left untreated or treated with 15 μg/mL of Sal4 IgA. At 5 h post-treatment, 100 µL of the culture from the top of the supernatant was collected and plated on LB agar containing X-gal to determine the relative composition of each strain. This portion of the culture was passaged, and the assay procedure was repeated the following day for a total of four rounds of treatment. Data represents the percentage of each strain (with the value for Δ oafA listed above the bar for the Sal4-treated groups) averaged from two biological replicates each with two technical replicates.

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: (A) Gene organization of fimW and neighboring genetic features, including the discontinued pseudogene stm14_0645 and the tRNA-encoding gene fimU . White boxes represent enriched spacers (32 nucleotides) identified in the screen analysis that target complementary sequences within fimW and stm14_0645 . For panels B and C, clearance of bacterial cells from the air-liquid interface of homogenous and mixed cultures of the indicated strains was quantified by plating colony forming units (CFUs) following 2 h of treatment with 15 μg/mL Sal4 IgA. (B) Data represents three biological replicates with error bars representing the standard deviation of the mean. Statistical significance was determined by ordinary two-way ANOVA with Šídák’s multiple comparison test. Asterisks (****) indicate p < 0.0001 and ns = not significant. (C) Data was obtained from four biological replicates with error bars representing the standard deviation of the mean. Statistical significance was determined by paired t-test (* indicates p < 0.05). (D) Percent composition of WT (SL174; lacZ + ) and Δ fimW (SL164; lacZ - ) recovered from the air-liquid interface as determined by blue-white screening of LB + X-gal plates. Values represent the average percentage of each strain from four biological replicates

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Standard Deviation, Comparison

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet:

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: (A) Recovered CFUs/mL of STm WT + pBAD24-EV (empty vector; EV), Δ fimW + EV, and Δ fimW + pBAD24- fimW (pFimW) cultures in the snow globe assay. (B) Quantification of mannose-sensitive yeast agglutination of the STm WT + EV, Δ fimW + EV, and Δ fimW + pFimW strains. (C) Recovered CFUs/mL of STm WT + EV, Δ fimW + EV, Δ fimA + EV, and Δ fimA + pFimW cultures in the snow globe assay. (D) Quantification of mannose-sensitive yeast agglutination of WT + EV, Δ fimW + EV, Δ fimA + EV, and Δ fimA + pFimW strains. For panels A and C, the indicated strains were grown to mid-log phase in the presence of 0.02% arabinose, washed in PBS, and either left untreated (circles) or treated with 15 μg/mL of Sal4 IgA (squares). After 2 h of treatment, the top of the supernatant was collected and plated on LB agar to measure CFUs. For panels B and D, the indicated strains were incubated statically for 48 h in LB containing 0.02% arabinose at 37 °C prior to centrifugation and resuspension in LB. Cultures were mixed with 10 mg/mL yeast in the presence (triangles) and absence (hexagons) of 3% mannose in a 12-well plate and the optical density of the wells at 600 nm (OD 600 ) was measured via spectrophotometry. The strains used are SL257, SL253, SL255, SL289, and SL291. For all panels, data was obtained from three biological replicates with error bars representing the standard deviation of the mean. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisks (**, ***, ****) indicate p < 0.01, p < 0.001, and p < 0.0001, respectively, and ns = not significant.

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Plasmid Preparation, Agglutination, Incubation, Centrifugation, Spectrophotometry, Standard Deviation

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: (A) Plates of 0.3% LB agar with and without 5.0 μg/mL Sal4 IgA were stab inoculated with 1.0 μL of overnight cultures of WT (SL174), Δ fimW (SL164), and Δ oafA (SL180) and then incubated at 37°C for 4.5 h. Plates were imaged and the diameter (mm) of bacterial migration was measured using Fiji. Data represents three biological experiments each averaged from three technical replicates. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisks (****) indicate p < 0.0001 and ns = not significant. (B) WT (SL174; lacZ + ) was mixed 1:1 with WT (SL239; lacZ - ), Δ fimW (SL164; lacZ - ), and Δ oafA (SL180; lacZ - ) and incubated for 15 min with 15 μg/mL of purified recombinant Sal4 IgA2m1 before addition to HeLa cell monolayers in 96-well microtiter plates. Plates were centrifuged at 1000 x g for 10 minutes (rotating the plate after 5 min) to promote bacteria-cell contact. After 1 h of incubation at 37 °C, monolayers were treated with 100 µg/mL gentamicin and incubated again for 1 h to eliminate extracellular bacteria. Cells were washed and lysed with 1% Triton X-100 diluted in Ca 2+ and Mg 2+ -free PBS and the resulting suspension was plated on LB containing X-gal to enable blue-white screening of CFUs. The competitive index [(%strain A output/%strain B output)/(%strain A input/%strain B input)] was calculated for each treatment group. Data represents three biological experiments each averaged from three technical replicates. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisks (*, **, ***) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, and ns = not significant.

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Incubation, Migration, Purification, Recombinant, Bacteria, Suspension

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: Mid-log phase cultures of the indicated strains were washed with LB, standardized to an OD 600 value of 1.0, and transferred to a 12-well polystyrene tissue culture plate. Cultures were left untreated (circles) or treated with 15 μg/mL Sal4 IgA (squares) at 23°C with shaking (200 rpm) for 1 h. The culture media was aspirated, and the biofilms were heat-fixed at 60°C for 1 h. Biofilms were stained with 0.1% crystal violet and washed with distilled water for 5 min, then the CV stain was solubilized with 30% acetic acid for 5 min. CV stain absorbance was quantified at Abs 570 . Data represents three biological experiments and error bars represent standard deviation of the mean. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisks (****) indicate p < 0.0001 and ns = not significant.

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Staining, Standard Deviation

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: Mid-log phase cultures of WT (SL174), Δ fimW (SL164), Δ oafA (SL180), and Δ flhC (SL202) were washed with LB, standardized to an OD600 value of 1.0 and transferred to borosilicate glass tubes. Cultures were mixed 1:1 with LB with (diamonds) and without (circles) 30 μg/mL Sal4 IgG and then incubated at 23°C with shaking (200 rpm) for 1 h (final concentration of 15 μg/mL Sal4). The culture media was aspirated, and the plate/tubes were heat-fixed at 60°C for 1 h. Biofilms were stained with 0.1% crystal violet and washed with distilled water for 5 min each, then the CV stain was solubilized with 30% acetic acid for 5 min. Dissolved CV was transferred to a 96-well plate prior to measurement of absorbance at 570 nm (Abs 570 ). (A) Representative images of the indicated strains 1 h p.t. (top) and with remaining CV stain after the final wash step (bottom). (B) Data represents three biological experiments and error bars represent standard deviation of the mean. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisks (**, ****) indicate p < 0.01 and p < 0.0001, respectively, and ns = not significant.

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Incubation, Concentration Assay, Staining, Standard Deviation

Journal: bioRxiv

Article Title: A Salmonella enterica serovar Typhimurium Genome-wide CRISPRi Screen Reveals a Role for Type 1 Fimbriae in Evasion of Antibody-Mediated Agglutination

doi: 10.1101/2024.12.16.628777

Figure Lengend Snippet: Sal4 IgA binding to the O5 antigen (dependent on expression of oafA ) induces a form of outer membrane stress that signals STm to transition from a planktonic to sessile state, either proximal to an abiotic surface or in suspension (step 1). Using flagellar-based motility, the bacteria swim towards an abiotic surface (step 2A) and collide into each other (step 2B). Fimbriae and Sal4-induced EPS production facilitate adhesion to the surface (step 3A). Hyperfimbriation, occurring through inhibition of fimW , enhances this step and reduces the extent of agglutination of STm in suspension (step 3B). Free-floating aggregates and surface-adhered microcolonies both bound by Sal4 IgA continue to produce EPS, resulting in the formation of biofilms (step 4AB).

Article Snippet: Liquid LB media with and without Sal4 IgA was combined with an equal volume of liquified 0.6% LB agar and then poured into a 100 x 15 mm square grid petri dish (ThermoFisher Scientific).

Techniques: Binding Assay, Expressing, Membrane, Suspension, Bacteria, Inhibition, Agglutination