Figure 7. Cbf1 negatively regulates competence.

• A
  Western blot of Cbf1‐3xFLAG levels after CSP stimulation. After 15 min, ˜ 2‐fold higher Cbf1‐3xFLAG levels could be detected, confirming cbf1 as a late‐competence gene as predicted by others (Peterson et al, 2004; Slager et al, 2019). CSP‐specific fold changes in Cbf1‐3xFLAG levels were calculated based on three independent experiments (Appendix Fig S7A). As control for the CSP induction, total RNA was extracted, run on an agarose gel, blotted, and probed for comCDE. A GAPDH‐specific antiserum and ethidium bromide staining were used as loading controls for the Western and northern blots, respectively.
• B
  Percentage of transformants using R6 and R6Δcbf1 in a spontaneous transformation assay. Bars represent the mean ± standard deviation percentage SmR CFU from 5 independent experiments. P‐value was calculated using an unpaired, two‐tailed t‐test.
• C
  Model of Cbf1‐dependent regulation of competence. The pneumococcal competence regulon is a quorum sensing system induced by activation of ComDE via CSP. Phosphorylated ComE induces transcription of the early competence gene loci comAB, comCDE, comX1, and comX2, leading to a positive feedback loop by production of ComC, which is processed into CSP and exported by ComAB. The paralogous alternative sigma factors ComX1 and ComX2 induce transcription of late‐competence gene loci, one of which is ccs50/cbf1 (Slager et al, 2019). Cbf1 is able to cleave, stabilize, and possibly activate csRNAs, which are induced by CiaRH, which in turn is activated by a variety of factors (Gómez‐Mejia et al, 2018). csRNAs are able to post‐transcriptionally downregulate comC. EC, extracellular. CM, cell membrane. IC, intracellular. CSP, competence‐stimulating peptide.

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