Part:BBa_K2273000

P_srfA: a ComA-dependent promoter of Bacillus subtilis

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.)

Usage and Biology

This promoter is controlled by the well-studied regulatory system for competence development in Bacillus subtilis (Figure 1). B. subtilis constantly secretes the ComX pheromone (BBa_K2273000), 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).

The most important promoter regulated by ComA and involved in competence development is the promoter of the srfA operon (P_srfA). This operon contains not only genes for the production of the antibiotic surfactin (e) but also for ComS (f), another small peptide that prevents the degradation of the autoregulated transcription factor ComK (g). 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_srfA (B) in different media. Wild type (WT, pink) and comX-knockout strains (ΔcomX, dark blue) contain a P_srfA 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_srfA (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_srfA- luxABCDEfusion. 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 iGEM team

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]