PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR PEPTIDE FACTOR THAT AFFECTS 2 DIFFERENT DEVELOPMENTAL PATHWAYS IN BACILLUS-SUBTILIS

We have purified and characterized an extracellular peptide factor tha t serves as a cell density signal for both competence development and sporulation in Bacillus subtilis. This competence and sporulation stim ulating factor (CSF) was purified from conditioned medium (culture sup ernatant) based on its ability to stimulate expression of srfA (comS) in cells at low cell density. CSF is a 5-amino-acid peptide, glu-arg-g ly-met-thr (ERGMT), that is, the carboxy-terminal 5 amino acids of the 40-amino-acid peptide encoded by phrC. No detectable CSF was produced in a phrC null mutant. The activity of chemically synthesized CSF (ER GMT) was virtually indistinguishable from that of CSF that was purifie d from culture supernatants. At relatively low concentrations (1-10 nM ), CSP stimulated expression of srfA, whereas high concentrations of C SF stimulated the ability of cells at low cell density to sporulate. S timulation of srfA expression by CSF requires the oligopeptide permeas e encoded by spo0K, a member of the ATP-binding-cassette family of tra nsporters, and the putative phosphatase encoded by rapC, the gene imme diately upstream of phrC. RapC was found to be a negative regulator of srfA expression, suggesting that the target of RapC is the transcript ion factor encoded by comA. We propose that CSF is transported into th e cell by the Spo0K oligopeptide permease and stimulates competence ge ne expression by inhibiting (either directly or indirectly) the RapC p hosphatase.