A New IS 4 Family Insertion Sequence, IS 4Bsu 1, Responsible for Genetic Instability of Poly-γ-Glutamic Acid Production in Bacillus subtilis

Abstract

ABSTRACT Certain Bacillus subtilis strains, such as B. subtilis ( natto ) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-γ-glutamate (γPGA). In B. subtilis ( natto ), γPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS 4Bsu1 , in the comP gene of a spontaneous γPGA-negative mutant of B. subtilis ( natto ) NAF4. IS 4Bsu1 (1,406 bp), the first IS discovered in B. subtilis , encodes a putative transposase (Tpase) with a predicted M r of 34,895 (374 residues) which displays similarity to the Tpases of IS 4 family members. Southern blot analyses have identified 6 to 11 copies of IS 4Bsu1 , among which 6 copies were at the same loci, in the chromosomes of B. subtilis ( natto ) strains, including NAF4, three commercial starters, and another three γPGA-producing B. subtilis ( natto ) strains. All of the eight spontaneous γPGA − mutants, which were derived from five independent NAF4 cultures, had a new additional IS 4Bsu1 copy in comP at six different positions within 600 bp of the 5′-terminal region. The target sites of IS 4Bsu1 were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS 4Bsu1 in mutant comP genes. These results indicate that IS 4Bsu1 transposes by the replicative mechanism, in contrast to other IS 4 members that use the conservative mechanism, and that most, if not all, of spontaneous γPGA − mutants appear to have resulted from the insertion of IS 4Bsu1 exclusively into comP . The presence of insertion hot spots in comP , which is essential for γPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous γPGA − mutation by IS 4Bsu1 in B. subtilis ( natto ).