Epoch spectrometer

We determined the hemolysis types of 11 strains (10 candidate mutant strains and a wild-type control) by streaking the bacterial cells on sheep blood agar plates. We selected a strain with a positive result from the blood agar plate assay for further quantitative investigation of hemolysis. We measured the hemolysis capability of each strain using the method previously described^{49 (link),50 (link)}. Briefly, the bacterial cells were cultured overnight, then sub-cultured in 10 mL of tryptic soy broth (TSB) for 3 h at 37 °C (optical density at 600 nm (OD₆₀₀) ≈ 0.6). After centrifugation of the bacterial culture at 13,000 rpm for 1 min at 4 °C, a 100-μL aliquot of the supernatant was mixed with 900 μL of 8% sheep blood, which was subsequently washed three times in phosphate-buffered saline (PBS). The resultant mixtures were incubated at 37 °C for 3 h, then centrifuged at 1,500 g for 10 min at 4 °C. We then measured the OD₄₅₀ for each strain using a BioTek Epoch spectrometer.

Total RNA was extracted using RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was stored at –80° C, and RNA sequencing and read alignments were conducted at ChunLab (Seoul, Korea). RNA quantity and quality were evaluated using an Epoch™ Spectrometer (BioTek, Winooski, VT, USA) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples met the following quality criteria: amount ≥1.1 μg, concentration ≥50 ng/μL, volume ≥20 μL, A260/280 ≥1.8, RNA integrity number ≥7, rRNA ratio (23S/16S or 28S/18S) ≥1.3.

Microbial genomic DNA was extracted from HRS and DRS samples using the FastDNA Spin Kit (MP Biomedicals, Irvine, CA, USA) and quantified using Epoch Spectrometer (Biotek, VT, USA). PCR amplification was performed using primers targeting V3 and V4 regions of 16S rRNA genes. The first round of amplification was carried out using primers 341F and 805R (Table S1) under the following conditions: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 5 min. Secondary amplification was performed to attach the Illumina NexTera barcodes using primers i5-F and i7-R (Table S1) under the same amplification conditions as described above; however, the number of amplification cycles was set to eight. The PCR products were separated by electrophoresis on 1% agarose gel and visualized using a Gel-Doc system (Bio-Rad, Hercules, CA, USA). Then, the PCR products were purified using the CleanPCR Kit (CleanNA, Waddinxveen, the Netherlands), and equal concentrations of the purified products were pooled together. Nontarget short fragments were removed using the CleanPCR Kit, and the quality and size of PCR products were assessed using the DNA 7500 chip on Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Pooled amplicons were sequenced at ChunLab, Inc. (Seoul, South Korea) using the Illumina MiSeq platform, according to the manufacturer’s instructions.