The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

Surfactin is a lipopeptide antibiotic produced by the cells of Bacillus subtilis ATCC 21332. A genetic locus responsible for surfactin production (sfp) was transferred by transformation from ATCC 21332 to JH642, a derivative of the standard B. subtilis 168. To study the sfp locus at the molecular level, a Tn917 insertion mutant that was blocked in surfactin production (sri) was isolated. The srf:Tn9l7 mutation was found to be closely linked to sfp, and both loci mapped by PBS1 phage transduction to the chromosomal region between arol and mtlB. These studies suggest that JH642, a strain which is not a producer of surfactin (genotypically sfpo), contains at least some of the genes encoding surfactin production. Expression of the srf gene(s) was examined in both sfp and sfpo cells by assaying l-galactosidase activity encoded by a promoterless lacZ gene that was fused to the sr.f:Tn917 insertion. In cells of both strains, sr-directed 0-galactosidase activity increased when cells entered the stationary phase of the growth curve, but the activity in sfp cells was higher than that in sfp°c ells. srf-lacZ expression was partially impaired by a mutation in spoOA. In sfpo cells, this dependence on the spoOA gene product could be entirely bypassed by an abrB suppressor mutation. In the sfp cells, the abrB mutation could not restore the defect conferred by the spoOA mutation. These data suggest that the sfp locus, which is responsible for surfactin production, alters the transcriptional regulation of srf in JH642 cells.

Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the ␣-subunit of RNA polymerase (RNAP). Here we report the discovery of an ''anti-␣'' factor Spx in Bacillus subtilis that blocks transcriptional activation by binding to the ␣-C-terminal domain, thereby interfering with the capacity of RNAP to respond to certain activator proteins. Spx disrupts complex formation between the activator proteins ResD and ComA and promoter-bound RNAP, and it does so by direct interaction with the ␣-subunit. ResD-and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATPdependent protease ClpXP. Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with ␣.Spx ͉ RNA polymerase ͉ ␣-subunit ͉ Bacillus subtilis ͉ transcriptional activation T ranscriptional activation in bacteria involves contacts between DNA-bound activators and promoter-bound RNA polymerase (RNAP). Most such interactions require the ␣-subunit of RNAP, which possesses several activator-interaction surfaces within its C-terminal domain (CTD) (1, 2). One such activator is ComA of the bacterium Bacillus subtilis (3-5), a response regulator, required for transcription of genes involved in the development of genetic competence (6). In response to high cell density, ComA becomes phosphorylated by interaction with its cognate histidine kinase, ComP, that is activated when it binds the pheromone ComX (7-9). ComA then activates the transcription initiation of the srf operon, which encodes the competence regulatory peptide ComS (10, 11). ComS serves to release the transcriptional activator ComK from its inhibitory complex composed of the proteins MecA and ClpCP (10-13), so that ComK can stimulate transcription of genes required for DNA uptake in competent cells (14). Interestingly, ComAdependent transcription of srf requires the ATP-dependent protease ClpXP (15, 16), which functions to eliminate the 15.4-kDa Spx protein (refs. 17 and 18; see Results). A mutation in clpX blocks ComA-activated transcription and has severe effects on growth and development (17). These pleiotropic effects of ClpXP absence can be suppressed either by the elimination of Spx or by missense mutations in the rpoA gene that encodes the RNAP ␣-subunit (refs. 16 and 17; see Results). This latter finding suggested that Spx exerts its negative effect on ComA-mediated transcription, and other transcriptional activation systems, by interaction with RNAP. A similar relationship between Spx and the ␣-subunit of RNAP was observed for ResD-activated transcription (see Results). ResD, like ComA, is a response regulator and transcriptional activator. It is part of the ResDE two-component signal transduction system that is required for the transcription of genes that are induced in response to oxygen limitation (19).In this article we show that Spx interferes with activatorstimulated transcription by interaction with the RNAP ␣-CTD, a mechanism of transcriptional repression ...