Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis

Abstract

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.

srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis

“…YT liquid medium (2x) and LB agar medium, prepared as described previously (20), were used for routine culture of B. subtilis and Escherichia coli. DS medium (20) or the S7 medium of Freese et al (3) was used for sporulation experiments. Surfactin production was assayed on blood agar plates (20).…”

“…DS medium (20) or the S7 medium of Freese et al (3) was used for sporulation experiments. Surfactin production was assayed on blood agar plates (20). Macrolide-lincosamidestreptogramin B-resistant (MLS') and chloramphenicol-resistant (Cmr) cells were selected as described in a previous report (20).…”

“…Surfactin production was assayed on blood agar plates (20). Macrolide-lincosamidestreptogramin B-resistant (MLS') and chloramphenicol-resistant (Cmr) cells were selected as described in a previous report (20). The other antibiotic concentrations used for selection of resistant transformant colonies were as follows: ampicillin, 25 ,ug/ml; neomycin, 10 ,ug/ml.…”

“…Three genes, sfp, sfA, and comA (previously called srfB) were identified and subsequently isolated (20,21). sfp is found in the surfactin-producing (SrfF, genotypically sfp) strain of B. subtilis.…”

“…sfp is found in the surfactin-producing (SrfF, genotypically sfp) strain of B. subtilis. When transferred by genetic transformation, sfp is necessary and sufficient to render cells of a nonproducing strain surfactin positive (20). All of the surfactin-nonproducing (Srf, genotypically sfp°) strains from B. subtilis examined thus far carry all of the genes that are required for surfactin production, with the exception of a functional sfp gene.…”