Taq ligase

Oligonucleotides for this study were synthesized by Integrated DNA Technologies (Coralville, IA) and are listed in Table S3. Restriction enzymes, T4 DNA ligase, Taq ligase, Q5 DNA polymerase, and T5 exonuclease, as well as PCR cleanup and gel extraction kits, were purchased from New England Biolabs (NEB). Plasmid and genomic DNA (gDNA) miniprep kits were purchased from Promega. Complementation plasmids were constructed using Gibson assembly as described previously (65 (link)). Plasmids used in this study are listed in Table S4. E. coli NEB 5α (NEB) or E. coli Mix and Go DH5α (Zymo) was used as a host for plasmid constructions, and all plasmids were confirmed by PCR or restriction digestion in addition to Sanger sequencing (Genewiz/Azenta). Plasmids from E. coli were introduced into S. aureus strain RN4220 by electroporation (66 (link)) prior to transfer into clinical isolates or directly into clinical isolates after passaging plasmids through E. coli strain IM08B (67 (link)). As needed, plasmids and mutant alleles conferring antibiotic resistance phenotypes were moved between S. aureus strains via ϕ11-mediated transduction or ϕ85-mediated transduction (68 (link)).

DNA was labeled according to commercial protocols using the IrysPrep Reagent Kit (BioNano Genomics, Inc). Specifically, 300 ng of purified genomic DNA was nicked with 7 U nicking endonuclease Nt.BspQI (New England BioLabs, NEB) at 37°C for two hours in NEB Buffer 3. The nicked DNA was labeled with a fluorescent-dUTP nucleotide analog using Taq polymerase (NEB) for one hour at 72°C. After labeling, the nicks were ligated with Taq ligase (NEB) in the presence of dNTPs. The backbone of fluorescently labeled DNA was stained with YOYO-1 (Invitrogen).

DNA was labelled following the IrysPrep Reagent Kit protocol (BioNano Genomics). Briefly, 900 ng of DNA was digested with 10 U of Nt.BspQI nicking endonuclease (New England BioLabs, Ipswich, MA) for 2 h at 37 °C. Nick digested DNA was then incubated for 1 h at 72 °C with fluorescently labelled dUTP and Taq Polymerase (New England BioLabs). Taq ligase (New England BioLabs) was used in the presence of dNTPs for ligation of nicks. DNA was counterstained with YOYO-1 (Life Technologies).

Yeast expression plasmids were purified from ~5ml saturated cultures using a Wizard^® DNA Purification System (Promega, Madison, WI, USA) and were transformed into competent yeast. The Gibson assembly principle [85 (link)] of incubating homologous PCR products with Taq ligase (NEB, Ipswich, MA, USA), T5 exonuclease (NEB) and Phusion polymerase (NEB) for 1 h at 50 °C, followed by plating on selective 2YT media, was used to create different fluorescently labelled Rpd3 expression constructs, which were confirmed by Sanger sequencing. Stable integrations of Ste3-GFP-DUb under control of the STE3 promoter were performed by linearising pCM850 with NsiI followed by ethanol precipitation and transformation into the various parental yeast strains. For strains with loxP flanked integrations, cassettes were excised using a modified TEF1-Cre expression system [86 (link)].

The DNA was labeled using the IrysPrep Reagent Kit (BioNano Genomics). Specifically, 300 ng of purified genomic DNA was nicked with 0.3 U of nicking endonuclease Nt.BspQI (New England BioLabs, NEB) at 37 °C for 2 h in buffer BNG3. The nicked DNA was labeled with a fluorescent-dUTP nucleotide analog using Taq polymerase (NEB) for 1 h at 72 °C. After labeling, the nicks were ligated with Taq ligase (NEB) in the presence of dNTPs. The backbone of fluorescently labeled DNA was counterstained with YOYO-1 (BioNano Genomics IrysPrep Reagent Kit).

All the point mutations in radA (radA_{C27A, K101A, K251A, R253A}) and recA (recA_K85A) were introduced by site-directed mutagenesis PCR on the recombinant plasmids, pET-radA and pKHS-recA accordingly. We used an adapted protocol from the Stratagene ‘QuickChange’ with only one mutator primer carrying the modified sequence for each mutation (Supplementary Table 1), and with both the Pfu turbo polymerase (Stratagene) and the TAQ ligase (New England Biolabs) in the PCR reaction.