Uncropped gel—nuclease data from: A nicking class 1 OLD family nuclease encoded by <em>Vibrio cholerae</em> inhibits virbiophage replication and is countered by a direct inhibitor

Bacteria are constantly threatened by their viral predators (phages), which has resulted in the development of defense systems for bacterial survival. One family of defense systems found widely across bacteria are OLD (for overcoming lysogenization defect) family nucleases. Despite recent discoveries regarding Class 2 and 4 OLD family nucleases and how phages overcome them, Class 1 OLD family nucleases warrant further study, as there has only been one anti-phage Class 1 OLD family nuclease described to date. Here, we identify the first bacteria-encoded Class 1 OLD family nuclease that exerts anti-phage activity: the Vibrio cholerae encoded Class 1 OLD family nuclease Vc OLD. We describe its disruption of the genome replication of the lytic vibriophage ICP1. Furthermore, we examine its in vitro activity, identifying Vc OLD as a DNA nickase. Finally, we identify the first direct inhibitor of a Class 1 OLD family nuclease, the ICP1-encoded Oad1. Our research further illuminates Class 1 OLD family nucleases’ role in phage defense and explores the dynamic arms race between V. cholerae and its predatory phage ICP1. Methods Nuclease assays with purified Vc OLD and Oad1 and bovine serum albumin (BSA) (Fisher BP1600) were carried out in 50-mM Tris–HCl, pH = 7.4, 50-mM NaCl, 10-mM MgCl2, 10-mM MnCl2, 10-mM CaCl2, 10NiCl2, and 10-mM ZnCl2. Frozen samples of all proteins were thawed and added to the buffer at varying concentrations. All nucleic acid substrates were eluted into H2O after nucleic acid collection. Double-stranded DNA (dsDNA) substrate was extracted from bacteria or phage following the procedure outlined in “Post-infection deep sequencing and coverage mapping.” Similarly, single-stranded DNA (ssDNA) (from M13 phage) was ordered from New England Biolabs (N4040S), extracted, and eluted into water. Total RNA was prepped from V. cholerae E7946 as described previously. Briefly, Bacterial strains were grown to an OD600 = 0.3, collected, and mixed 1:1 with ice-cold methanol. Methanol-treated samples were pelleted at 7000 × g at 4°C, and the resulting pellets were washed in ice-cold 1× phosphate-buffered saline. The washed pellets were resuspended in 200 μl TRI Reagent (Millipore/Sigma) and incubated for 5 min at room temperature. The samples were mixed with 40 μl chloroform, vortexed, and incubated for 10 min at room temperature. The chloroform-treated samples were then centrifuged at 12 000 × g for 10 min at 4°C. Following centrifugation, the upper (aqueous) phase was collected, promptly mixed with 110 μl 2-propanol and 11 μl pH 6.2 3-M sodium acetate, and mixed vigorously. The samples were centrifuged at 12 000 × g for 15 min at 4°C, and the pellets were washed twice with 75% ethanol. The washed pellets were incubated at 65°C for 3 min to evaporate residual ethanol. The RNA was resuspended in 10–30 μl of diethyl dicarbonate water, and RNA concentration were analyzed by NanoDrop. Plasmid DNA substrate, a pUC19 derivative, was prepped using the NEB Monarch Plasmid Miniprep Kit. All DNA substrates were added at 100 ng per 20 μl reaction. RNA was loaded at 500 ng per 20 μl reaction. Reactions containing DNA substrate were incubated at 37°C for 3 h, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. Reactions containing RNA substrate were incubated at 37°C for 30 min, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. To generate linear or nicked controls, 100 ng of vector was digested in 1× CutSmart Buffer for 1 h at 37°C with 10 U of BamHI-HF (NEB) or 10 U of Nb.BtsI (NEB) in 20 μl reactions, respectively, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. The entire reaction volume was loaded and run on a 0.8% agarose gel stained with GelRed.