ISOLATION OF A NEW SURFACTIN PRODUCER BACILLUS-PUMILUS A-1, AND CLONING AND NUCLEOTIDE-SEQUENCE OF THE REGULATOR GENE, PSF-1

Two bacteria (A-1 and B-1) which exhibited large emulsified halos around their colonies on oil-L-agar plates were isolated. These bacteria produced the same biosurfactant, surfactin. Strain A-1 (Psf+: surfactin producing) was identified as Bacillus pumilus and could be transformed with plasmid DNA using an electroporation method. Several surfactin non-producing (Psf-) mutants were obtained by chemical mutagenesis of B. pumilus A-1. By using them as host cells and pC194 as a vector plasmid, we cloned DNA fragments which complemented Psf- alleles. One of them (2 kb HindIII fragment) could transform Bacillus subtilis MI113 (a derivative of Marburg strain 168 and Psf-) to a Psf+ cell as well. Nucleotide sequence of the 2 kb fragment was determined and three large open reading frames (ORF1, 2, 3) were found. Deletion analysis of the recombinant plasmid indicated that ORF3 (699 bp, 233 amino acid residues) is essential for surfactin production. The gene was designated as psf-1 and a 25 kDa translation product was detected. The nucleotide sequence of psf-1 exhibited high homology with an unknown open reading frame, orfX, upstream to the gramicidin S biosynthesis operon of Bacillus brevis. It is suggested that the deduced gene products of unknown orfX in B. brevis and psf-1 in B. pumilus may share the same function.