Part:BBa_K2273002

P_rapF: a ComA-dependent promoter of Bacillus subtilis

Usage and Biology

Figure 1: The regulatory system for competence development in Bacillus subtilis. The development of genetic competence is stimulated by quorum sensing due to the secretion of the ComX pheromone and its accumulation in the surrounding medium. (Modified from: Jung et al.)

This promoter is under the control of the well-studied regulatory system for competence development in Bacillus subtilis (Figure 1). B. subtilis constantly secretes the ComX pheromone (BBa_K2273003), a 9- to 10-amino acid oligopeptide, as a signalling molecule (a). By rising cell-density, the ComX-concentration in the surrounding medium increases until it reaches a threshold and activates ComP, a membrane-spanning protein kinase (b). This mechanism is called quorum sensing. The kinase reacts to the accumulation of ComX by phosphorylating the response regulator ComA (c) which then works as a transcription factor by binding to several promoters and enhancing their activity (d).
According to literature the promoter P_rapA is regulated by ComA. Its sequence contains a canonical and non-canonical binding site of ComA.
Since the promoter is not only dependent on quorum sensing but also on nutritional conditions, we evaluated the influence of different media (Figure 2). We tested full medium (LB) as well as minimal media (MNGE, MCSE).

Figure 2: Growth (A) and promoter activity of P_rapA (B) in different media. Wild type (WT, pink) and comX-knockout strains (ΔcomX, dark blue) contain a P_rapA-luxABCDE fusion.

Furthermore, we studied the influence of ComX-overproduction on the promoter activity by integrating an inducible copy of comX.

Growth (A) and promoter activity of P_rapA (B) in dependence of xylose-induction. Wild type (WT, pink and light blue) and comX-knockout strain (ΔcomX, black and dark blue) contain a P_rapA-luxABCDE fusion. After 1 hour of incubation (dashed line) the cultures were induced with 1% xylose (light blue and black) or with distilled water (dH2O, pink and dark blue).

The promoter was created and used by TU_Dresden 2017. All assays have been conducted with B. subtilis W168 carrying the promoter fused to the lux operon.Other promoters that haven been evaluated regarding their ComX-dependent activity:

PsrfA	BBa_K2273000
PrapA	BBa_K2273001
PrapF	BBa_K2273002
PcomG	BBa_K2273012
PcomK mut	BBa_K2273013

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]