Journal: bioRxiv

Article Title: Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

doi: 10.1101/2023.01.30.526351

Figure Lengend Snippet: DNA binding by PlzA is c-di-GMP dependent. ( a ) EMSA with increasing concentrations of PlzA WT showing binding to glpFKD (−219) DNA. The mixture was incubated for 10 min before electrophoresis. ( b ) The same EMSA mixture as in ( a ) but with a final concentration of 2.5 ng/μL of the nonspecific competitor poly-dI-dC. Protein was incubated with poly-dI-dC for 5 min prior to the addition of labeled glpFKD (−219) probe. ( c ) The same EMSA mixture as in ( b ) but supplemented with 100 μM c-di-GMP. Protein was incubated with c-di-GMP for 5 min prior to the addition of any nucleic acids. Arrows indicate PlzA- glpFKD (−219) complexes. Lane 1 in ( a-c ) is the probe only control, containing 50 nM of probe. Protein concentrations are as follows in each subsequent lane: Lane 2: 0.5 μM, Lane 3: 1 μM, Lane 4: 2.5 μM, Lane 5: 5 μM, Lane 6: 7.5 μM, and Lane 7: 10 μM. ( d ), ( e ), ( f ) EMSA experiments as in ( a ), ( b ), and ( c ), respectively, but performed with the mutant PlzA RD-RD protein. All conditions were performed identically as with PlzA WT . Each panel is a representative gel from separate EMSAs which were performed in triplicate (PlzA WT ) or duplicate (PlzA RD-RD ).

Article Snippet: Prior to the addition of labeled probe, the non-specific competitor poly-dI-dC (Roche) was added to the EMSA reactions at a final concentration of 2.5 ng/μL, and the reaction mix was allowed to incubate another 5 min ( ).

Techniques: Binding Assay, Incubation, Electrophoresis, Concentration Assay, Labeling, Control, Mutagenesis

Journal: bioRxiv

Article Title: Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

doi: 10.1101/2023.01.30.526351

Figure Lengend Snippet: Levels of c-di-GMP alter DNA binding affinity of PlzA WT . ( a ) A diagram of the generated DNA probe, designated as glpFKD (−7/+35), corresponding to a 42 bp sequence adjacent to the −10 site and proceeding 35 bp into the 5’ UTR. An RNA probe was made corresponding to only nucleotides that are within the 5 ‘UTR which was designated as glpFKD (UTR) RNA. Probes were conjugated to a fluorescent molecule for the detection of binding in EMSAs. ( b ) EMSAs were performed with increasing protein concentrations of PlzA WT, and 10 nM fluorescently labeled probe at varying c-di-GMP concentrations (0, equimolar, 10, 25, 50, or 100 μM). For each EMSA shown, the indicated c-di-GMP concentration was held constant in all lanes. For each EMSA shown, lanes 1 and 7 are probe-only controls. Protein concentrations were as follows: Lane 2– 1 μM, Lane 3– 2.5 μM, Lane 4– 3.5 μM, Lane 5– 5 μM, Lane 6– 7.5 μM. A final concentration of 2.5 ng/μL of the nonspecific competitor poly-dI-dC was added to each lane. Arrows indicate higher-order complexes formed as protein concentration increases. A representative gel for each tested c-di-GMP concentration is shown from triplicate EMSAs. ( c ) Triplicate EMSAs were quantitated by densitometry to determine the K d(app) of the PlzA WT - glpFKD (−7/35+) interaction by nonlinear regression analysis using the one-site specific binding setting in GraphPad Prism. Errors bars represent one standard deviation (SD). ( d ) Bar graph displaying the calculated K d(app) ± the standard error of the mean (SEM). The K d(app) values from three EMSAs were analyzed by a Welch ANOVA with Dunnett’s T3 multiple comparisons test via GraphPad Prism. Adjusted P-values are indicated as follows: ns= p >0.05, *= p ≤0.05, and ***= p ≤0.001. Comparisons between 10, 25, 50, and 100 μM c-di-GMP are not indicated on the graph as they were not significant. ( e ) A similar EMSA experiment was performed as in but with PlzA RD-RD and either 100 (lanes 1–6) or 0 (lanes 7–12) μM of c-di-GMP supplemented. No binding is observed.

Article Snippet: Prior to the addition of labeled probe, the non-specific competitor poly-dI-dC (Roche) was added to the EMSA reactions at a final concentration of 2.5 ng/μL, and the reaction mix was allowed to incubate another 5 min ( ).

Techniques: Binding Assay, Generated, Sequencing, Labeling, Concentration Assay, Protein Concentration, Standard Deviation

Journal: bioRxiv

Article Title: Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

doi: 10.1101/2023.01.30.526351

Figure Lengend Snippet: Competition EMSAs with mutagenized glpFKD (−7/35+) competitors. ( a ) A diagram of the generated unlabeled, mutagenized glpFKD (−7/+35) competitors. Comp 1 (C1) is the unlabeled, specific glpFKD (−7/+35) competitor. Introduced mutations are indicated in red for each mutant competitor. The transcriptional start site is the nucleotide labeled as blue. Competition EMSAs were performed with the various mutant competitors and the glpFKD (7/35+) probe. ( b ) PlzA WT , c-di-GMP, poly-dI-dC, and glpFKD (−7/35+) probe concentrations were held constant at 2.5 μM , 100 μM, 2.5 ng/μL, and 10 nM respectively. Lane 1 is the probe-only control, while lane 2 is the probe + protein only control. Lanes 3–5: 250, 500, & 1000x molar excess (relative to the probe) of comp 1 (unlabeled, glpFKD (−7/35+). Lanes 6–8: Lanes 3–5: 250, 500, & 1000x mut 1 competitor. Lanes 6–8: Lanes 9–11: 250, 500, & 1000x mut 2 competitor. ( c ) Concentrations for protein, effector, nonspecific competitor, and probe as in ( a ). Lane 1 is the probe-only control, while lane 2 is the probe + protein only control. Lane 3 is the specific competitor control with 1000x of comp 1. Lanes 4–6: 250, 500, & 1000x mut 4 competitor. Lanes 7–9: 250, 500, & 1000x mut 3 competitor. Lanes 10–12: 250, 500, & 1000x mut 5 competitor. ( d ) PlzA WT , c-di-GMP, poly-dI-dC, and glpFKD (−7/35+) probe concentrations were held constant at 3.5 μM , 100 μM, 2.5 ng/μL, and 10 nM respectively. Lane 1 is the probe-only control, while lane 2 is the probe + protein only control. Lanes 3–5: 250, 500, & 1000x molar excess (relative to the probe) of comp 1 (unlabeled, glpFKD (−7/35+). Lanes 6–8: Lanes 3–5: 250, 500, & 1000x mut 6 competitor. The black line denotes irrelevant lanes that were removed from the same gel. ( e ) PlzA WT , c-di-GMP, poly-dI-dC, and glpFKD (−7/35+) probe concentrations were held constant at 2.0 μM , 100 μM, 2.5 ng/μL, and 10 nM respectively. Lane 1 is the probe-only control, while lane 2 is the probe + protein only control. Lane 3 is the specific competitor control with 1000x of comp 1. Lanes 4–6: 250, 500, & 1000x mut 7 competitor. Lanes 7–9: 250, 500, & 1000x mut 8 competitor. Lanes 10–12: 250, 500, & 1000x mut 9 competitor.

Article Snippet: Prior to the addition of labeled probe, the non-specific competitor poly-dI-dC (Roche) was added to the EMSA reactions at a final concentration of 2.5 ng/μL, and the reaction mix was allowed to incubate another 5 min ( ).

Techniques: Generated, Mutagenesis, Labeling, Control

Journal: bioRxiv

Article Title: Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

doi: 10.1101/2023.01.30.526351

Figure Lengend Snippet: Competition EMSAs with truncated glpFKD (−7/35+) competitors. ( a ) A diagram of the generated unlabeled, truncated glpFKD (−7/+35) competitors. Comp 1 (C1) is the unlabeled, specific glpFKD (−7/+35) competitor. Competitors 2, 3, and 4 are unlabeled glpFKD (−7/+35) truncated at the 5’ end by either 9, 12, or 15 bases respectively. The transcriptional start site is the nucleotide labeled as blue. ( b ) PlzA WT , c-di-GMP, poly-dI-dC, and glpFKD (−7/35+) probe concentrations were held constant at 2.0 μM , 100 μM, 2.5 ng/μL, and 10 nM respectively. Lane 1 is the probe-only control, while lane 2 is the probe + protein only control. Lane 3 is the specific competitor control with 1000x of comp 1. Lanes 4–6: 250, 500, & 1000x comp 2 competitor. Lanes 7–9: 250, 500, & 1000x comp 3 competitor. Lanes 10–12: 250, 500, & 1000x comp 4 competitor. A representative gel is shown from two independently performed experiments.

Article Snippet: Prior to the addition of labeled probe, the non-specific competitor poly-dI-dC (Roche) was added to the EMSA reactions at a final concentration of 2.5 ng/μL, and the reaction mix was allowed to incubate another 5 min ( ).

Techniques: Generated, Labeling, Control

Journal: bioRxiv

Article Title: Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

doi: 10.1101/2023.01.30.526351

Figure Lengend Snippet: Oligonucleotides used in this study.

Article Snippet: Prior to the addition of labeled probe, the non-specific competitor poly-dI-dC (Roche) was added to the EMSA reactions at a final concentration of 2.5 ng/μL, and the reaction mix was allowed to incubate another 5 min ( ).

Techniques: Sequencing, Clone Assay, Mutagenesis