Materials and Methods s5

Materials and Methods

Plasmids and strains:

Pseudo-origin sequences were generated in silico using Geneious Pro 5.0.3, synthesized by Eurofins, MWG Operon, and arrived cloned into the pCR2.1 topo vector. Plasmids for the transformation assay were generated as follows. Pseudo-origins from these vectors were pcr amplified from the pCR2.1 constructs using PFU Ultra II Fusion HS DNA Polymerase (Agilent Technologies) and various pseudo-ori 5’ Xma fuse and pseudo-ori 3’ Xho fuse primers found in Table S2. These pcr fragments were then purified using a Qiagen PCR purification kit and cloned into pYB199 (14) digested with XhoI, XmaI (NEB) via Gibson assembly (48).

Plasmids for EMSA probes were generated as follows. Pseudo-origin fragments used were pcr amplified from the pCR2.1 constructs using PFU Ultra II Fusion HS DNA Polymerase (Agilent Technologies) and primers from Table S2. The Inc-11-mer and Inc-12-mer fragments were pcr amplified using genomic DNA from V. cholerae strain N16961 using PFU Ultra II Fusion HS DNA Polymerase (Agilent Technologies) and primers from Table S2. These fragments were cloned into the pBlueScript vector by incubation with SmaI and T4 DNA ligase at room temperature overnight.

All clones were verified by sequencing by Genewiz.

Electrophoretic mobility shift assay:

Mini-preps from Dam+ or Dam- E. coli were treated with XbaI, XhoI, and Calf Intestinal Phosphatase (NEB) at 37oC overnight. The EMSA probes were gel purified and cleaned with a GE Illustra microspin G-50 column. DNA equivalent to 12.5 pmol of ends was incubated with 12.5 pmol ATP gamma P32 and T4 Polynucleotide Kinase at 37°C overnight. Labeled fragments were cleaned with GE Illustra microspin G-50 columns, phenol-chloroform extracted, and ethanol precipitated. Labeled probes were resuspended to a concentration of 20ng/uL in Tris-EDTA pH 8.0. 0.1 nM of probe was incubated in 20uL reactions with amounts of RctB indicated in figures for 10’ at room temperature. The Binding Buffer composition was 20mM Tris-Cl pH 7.5, 1mM EDTA, 150mM NaCl, 100ug/mL BSA, 12.5ug/mL poly dI-dC (Sigma-Aldrich). After the 10’ incubation, 5uL of 1x Binding Buffer with 50% glycerol was added to the reaction, to a final concentration of 10% glycerol. 20uL of the 25uL binding reaction was loaded onto a 6% DNA retardation gel (Invitrogen) and run in 0.5x TBE at 425V, 15mA, and 25.0W for 30’-40’ depending on the length of the DNA fragment. Gels were dried down onto filter paper with a Biorad Model 583 gel dryer and Hydrotech vacuum pump. Gels were exposed to a Kodak storage Phosphor screen and visualized with a FujiFilm FLA-5100 imager. Bands were quantitated with MultiGuage V3.1 image analysis software. Graphs and binding characteristics (Kd’s) were generated from the quantitation results using GraphPad Prism 6 software and fitting the data with the non-linear standard sigmoidal curve, Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*Hill Slope)).

Transformation Efficiency Assay:

The pseudo-origins and kanamycin cassettes were obtained by digestion of the pYB199-pseudo-origin constructs with XbaI and ApaI and gel purification of the ~1.9 kb fragment with a Qiagen gel purification kit. The fragments were cleaned with a GE Illustra Microspin G-50 column and 200ng of each pseudo-origin fragment was ligated in a 20uL ligation reaction using T4 DNA ligase (NEB) at 16°C overnight. The ligations were dialyzed against diH2O for 15’ using Millipore MF Nitrocellulose membrane filters with a 0.05 um pore size. 3uL of the dialyzed ligations were transformed into 100uL of electro-competent DH5a E. coli harboring either pGZ1119, pYB285, or pYB296. Electro-competent E. coli were made by growing cells in LB with chloramphenicol (Cam, 20ug/mL) and Isopropyl-B-D-1-thiogalactopyranoside (IPTG, 100uM) at 37oC with shaking to an OD600 between 0.6-0.8. The transformations were outgrown in 1mL SOC media for 1 hour at 37ºC with shaking. 100uL and 1mL of the transformations were plated onto LB Cam (20ug/mL) Kanamycin (Kan, 50mg/mL) IPTG (100uM) 1% agar plates and incubated at 37ºC overnight, then room temperature overnight. 100ng of pYB190 was also transformed into each strain to normalize for transformation efficiency. The number of transformants for each pseudo-origin was divided by the number of colonies for pYB190 and then the ratio for each mutant was divided by the ratio for the wild-type oriCII to obtain the results shown in Figure 4. Averages and standard errors of the mean were generated from 3 independent experiments.

Ori-seq:

The oriCII mutant library was generated using the following steps. oriCII-min DNA fragments were generated by mutational pcr using the GeneMorph II EZclone domain mutagenesis kit (Agilent) on pYB199-oriCII-wt and primers pseudo-ori mut 5’ Xma fuse and pseudo-ori mut 3’ Xho fuse. We used 50ng of pYB199 as template and did 50 amplification rounds, in an attempt to obtain ~10-15 mutations per kb. The Kan resistance cassette was pcr amplified from pYB199 using PFU Ultra II Fusion HS DNA Polymerase (Agilent Technologies) and primers kan 5’ Xma fuse and kan 3’ Xho fuse. The pcr fragments were purified with a Qiagen pcr purification kit and ligated using Gibson assembly (48). The assembled mutant library was transformed into electro-competent DH5a E. coli cells harboring either pYB285 or pYB296 made as above. Transformants were outgrown in SOC for 1 hour at 37°C with shaking and then plated onto LB Cam (20ug/mL) Kan (50ug/mL) IPTG (100uM) 1% agar plates and placed at 37°C overnight. A second round of Gibson assembly and transformation was done and colonies from both experiments were pooled based on strain. RctB wt (pYB285) had approximately 50,000 and 44,000 colonies, for a total of 94,000. RctB deltaC159 (pYB296) had approximately 38,000 and 45,000 colonies, for a total of 83,000.

Plasmid DNA was prepped using 1/8th of each strain pool using a Qiagen mini-prep kit. The entirety of both mini-preps was digested with BstAPI, BssHII, and NaeI (NEB). The digests were run on 1.5% agarose gels and the 531 bp fragment was excised and purified using a Qiagen gel purification kit and cleaned with GE Illustra microspin G-50 columns. The mutant libraries were then prepared for sequencing using the Nextera XT DNA Sample preparation Kit and indices from the Nextera XT Index Kit (Illumina). The number of reads per uL of each library was quantitated by qPCR against a standard curve and the amount of DNA to obtain 12 million reads for each strain was sequenced using a 600 cycle MiSeq Reagent Kit v3 and a MiSeq sequencer (Illumina).

The reads were trimmed and evaluated for quality score with CLC Genomics Workbench (CLCbio) and then mapped to the wild type oriCII-min sequence of V. cholerae (bases 514-887 of oriCII, Fig. 1) using the Bowtie aligner (49). A custom Python script was used to analyze the number of mismatches and total reads for each base, resulting in the average mutation frequency at each base. The mutation frequency at every base was averaged to obtain the average mutation frequency per base across all the mutant oriCII-min fragments sequenced. A comparison of each individual base mutation frequency versus the average mutation frequency was done and this analysis visualized with Artemis, Release 15.0.0 (Welcome Trust Sanger Institute).

Comparative Seq:

The oriCII-min (bases 514-887 of oriCII, Fig. 1) sequence of V. cholerae was used to perform a standard nucleotide BLAST on the NCBI website against Vibrionaceae optimized for more dissimilar sequences (discontinuous megablast). This resulted in a comparison against 28 Vibrionaceae species, which are listed in Table S3. The total number of times an individual base was present as well as the total number of mismatches for that base across the 28 species was analyzed using a custom Python script. The mutation frequency at every base was averaged to obtain the average mutation frequency per base across all the mutant oriCII-min fragments sequenced. A comparison of each individual base mutation frequency versus the average mutation frequency was done and this analysis visualized with Artemis, Release 15.0.0 (Welcome Trust Sanger Institute).

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