Genome Sequencing of Suppressor Mutants

Strains IU5844, IU6441, and IU6442 containing spontaneous suppressor mutations that allowed growth of a D39 Δcps ΔgpsB deletion mutant were isolated as described in Results (see Table 2). Overnight cultures still in exponential phase were diluted into 5 ml of BHI broth to an OD₆₂₀ ≈ 0.01 and grown to an OD₆₂₀ ≈ 0.3 to 0.4. Cells were collected by centrifugation (10,000×g for 10 min at 25 °C). Genomic DNA was purified using a MasterPure Gram-positive DNA purification kit (Epicenter Biotechnologies) according to the manufacturer's protocol. DNA library construction, Illumina MiSeq DNA sequencing, and bioinformatics analyses were performed as described previously (Tsui et al., 2016 (link)). Visual inspection of the coverage data revealed gaps in genomic sequences corresponding to the expected Δcps Δ[cps2A-cps2H] and ΔgpsB deletion mutations and to the Δ[spd_1026–spd_1037] and Δ[spd_1029–spd_1037] deletions and adjacent chromosomal duplications in strains IU5845 and IU6441, respectively (see Table 2; Fig. S3). The structures of the rearrangements were verified by assembling the genome sequences using newbler (version 2.9). The resulting contig graph was analyzed to determine the organization of the duplications/deletions (see Fig. S3B).