S1 File - The conserved transcription factor PrlP modulates colonization and pathogenicity of Streptococcus suis in response to environmental stress

S1 Fig. Screening for mutants with growth defects under acidic condition. (A) Screening for key genes involved in S. suis proliferation under acidic conditions. The S. suis transposon mutant library is cultured under acidic conditions to identify essential genes. The selected mutants are linked to sequencing adaptors, amplified, and then subjected to high-throughput sequencing. Target genes are knocked out and complemented, followed by growth curve analysis to validate their impact on bacterial growth. (B) Screening for S. suis mutants with growth defects under acidic conditions. Transposon mutants were cultured in TSB medium adjusted to pH 6.0 and pH 7.0 for 8 hours, and OD600 values were recorded to calculate the growth ratio (pH 6.0/ pH 7.0) as an indicator of acid tolerance. This figure shows the screening data from one representative 96-well plate containing prlP group mutants (as an example subset of the overall library). Several mutants exhibited impaired growth under acidic conditions, among which strain No. 53 consistently showed the most pronounced and reproducible growth defect across independent experiments (highlighted in red) and was therefore selected for further analysis. Data are presented as mean ± SD, and statistical analysis was performed using two-tailed unpaired t-tests (n = 3). S2 Fig. Construct prlP gene knockout and complementation strains. (A) The confirmation of ΔprlP with PCR reaction. The pairs of primers included the external primers (PrlP-L-F/R-R) and the internal primers (PrlP-F/R). S3 Fig. Functional conservation of PrlP in the reference S. suis strain P1/7. (A) Growth curves of wild-type P1/7 and the isogenic ΔprlP mutant in TSB at 37°C. The ΔprlP strain showed significantly impaired growth compared to the wild type. (B) Growth performance under different pH conditions (pH 6.0, 7.0, and 8.0) after 8 h of incubation. The ΔprlP mutant exhibited reduced growth under acidic conditions and mild impairment at pH 7.0, similar to the phenotype observed in the SC19 background. (C) Stress tolerance assays under hyperosmotic (1.5% NaCl) and heat (42°C) stress conditions. The ΔprlP mutant was more sensitive to both stressors compared to the wild-type P1/7. Data are presented as mean ± SD from three independent experiments, with statistical significance determined by two-tailed unpaired t-tests. S4 Fig. Construct PrlP domain-specific knockout and complementation strains. (A) The confirmation of prlP-ΔN and prlP-ΔC with PCR reaction. The pairs of primers included the primers (PrlP-ΔN-L-F/R-R) and primers (PrlP-ΔC-L-F/R-R). S5 Fig. Construction and verification of the prlP-ΔCself-cleavage mutants. PCR confirmation of the prlP-ΔCself-cleavage deletion mutant using external primer pair PrlP-Cself-cleavage-EX-F/R. S6 Fig. Construct double-knockout strains for potential PrlP-regulated genes. (A) The confirmation of ΔprlP with PCR reaction. The confirmation of ΔprlP with PCR reaction. The pairs of primers included the primers (PrlP-ΔN-L-F/R-R) and primers (PrlP-ΔC-L-F/R-R). S7 Fig. Expression and purification analysis of PrlP. (A) SDS-PAGE and Western blot analysis of the expressed and purified PrlP under denaturing conditions. The gel shows the presence of PrlP at the expected molecular weight. S8 Fig. Structural prediction of B9H01_08740. Domain architecture of the B9H01_08740-encoded protein. The protein consists of 1,122 amino acids and contains conserved motifs corresponding to the YebA superfamily (residues 222–350) and the ribonuclease E superfamily (residues 532–1002), as predicted by domain analysis. (RAR)