RopB represses the transcription of speB in the absence of SIP in group A Streptococcus

(A) The expression of speB in the wild-type strain and its pepO mutant, covR mutant, and ∆covR/∆pepO mutant. (B, C) Transcription of speB and the expression of SpeB in the covR mutant, ∆covR/∆ropB mutant, and SIP*/∆covR mutant. (D, E) Transcription of speB and expression of SpeB in the ∆covR/∆ropB mutant, SIP*/∆covR mutant, and SIP*/∆covR/∆ropB mutant. Culture supernatant was used for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB. Bacterial RNA was extracted for real-time quantitative PCR (qPCR) analysis. The expression of speB was normalized to that of gyrA. *P < 0.05.

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(A) The phosphorylation level of CovR in the wild-type strain (Wt), CovS kinase-inactivated (CovS_H280A) mutant, and CovS phosphatase-inactivated (CovS_T284A) mutant. The ∆covS mutant (the mutant that cannot phosphorylate CovR) and the covR mutant (∆covR) were used as experimental controls. The total protein is used as the internal loading control. (B) The expression of PepO in the wild-type strain, its pepO mutant (∆pepO), ∆covS mutant, and the ∆covS/∆pepO mutant. The lower panel shows the total protein as the internal loading control. (C) The transcription of speB in the ∆covS mutant and ∆covS/∆pepO mutant under the synthetic SIP and scramble peptide (SCRA) treatments. (D) The transcription of ropB in the wild-type strain, CovS_H280A mutant, and CovS_T284A mutant. (E) The expression of SpeB in the wild-type strain, CovS_H280A mutant, CovS_T284A mutant, and their ropB mutants. (F) The expression of speB in the CovS_H280A mutant and its ropB mutant (CovS_H280A/∆ropB). (G) The transcription of speB in the CovR D53A substitution mutant (CovR_D53A) and its ropB mutant (CovR_D53A/∆ropB). Culture supernatant was used for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB. Bacterial RNA was extracted for real-time quantitative PCR (qPCR) analysis. The expression of ropB and speB was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

(A, B) The genes those were differentially expressed in (A) SIP*/∆ropB mutant versus the wild-type strain (Wt) and (B) SIP*/∆ropB mutant versus the SIP* mutant are visualized by the volcano plot. Blue circles and red circles indicate the down-regulated and up-regulated genes, respectively, in the (A) SIP*/∆ropB mutant compared with that of the wild-type strain and (B) SIP*/∆ropB mutant compared with that of the SIP* mutant (P < 0.05). The solid circles indicate that the expression difference is statistically significant (adjusted P-value, q value < 0.05). (C) Schematic representation of the speB, spi, and prsA genes (arrows). The speB and its co-transcripts (dashed lines) and the location of primers (Primer-1–Primer-3) used for detecting speB and its co-transcripts are indicated. The genes and their annotations are indicated according to MGAS5005 (NCBI Accession: CP000017.2; the open arrows) and SF370 (NCBI Accession: NC_002737.2; the gray arrow). (D) The expression of speB and its co-transcripts in the wild-type strain, the ropB mutant, SIP* mutant, and SIP*/∆ropB mutants detected by Primer-1–Primer-3. Bacterial RNA was extracted for sequencing and real-time quantitative PCR (qPCR) analyses. The expression of the target transcript was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

(A, B) Expression of (A) M5005_Spy1176 (negatively regulated by RopB-SIP) and (B) M5005_1416 and M5005_Spy1426 (positively regulated by RopB-SIP) in A20, the SIP* mutant, and SIP*/∆ropB mutant in the early stationary phase of growth (O.D.₆₀₀ = 1.0). The upper and lower panels of (A, B) show the results from RNA-seq analysis and real-time quantitative PCR (qPCR) analysis, respectively. (C, D, E) Expression of (C) ropB and speB, (D) M5005_Spy1176, and (E) M5005_1416 and M5005_Spy1426 in A20, the SIP* mutant, and SIP*/∆ropB mutant under neutral (pH 7.5) and acidic (pH 6.0) conditions. RNAs were extracted for qPCR analysis. The expression of target genes was normalized to that of gyrA. bdl, below detection limit. *P < 0.05.

Source data are available for this figure.

The expression of ropB and pepO are repressed by CovR. Although the up-regulated PepO would degrade SIP in the covR mutant, the effect of PepO degradation could be compensated by the derepression of ropB and SIP, and the SIP-bound RopB (RopB-SIP) could activate speB transcription. In the covS mutant, the expression of pepO is up-regulated, whereas that of ropB is repressed by the non-phosphorylated CovR. Therefore, the repression of speB in the covS could be mediated by the PepO-dependent SIP degradation and the SIP-free RopB (apo-RopB)-dependent transcriptional repression.