A New IS4 Family Insertion Sequence, IS4Bsu1, Responsible for Genetic Instability of Poly-γ-Glutamic Acid Production in Bacillus subtilis

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 IS4Bsu1, in the comP gene of a spontaneous γPGA-negative mutant of B. subtilis (natto) NAF4. IS4Bsu1 (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 IS4 family members. Southern blot analyses have identified 6 to 11 copies of IS4Bsu1, 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 IS4Bsu1 copy in comP at six different positions within 600 bp of the 5′-terminal region. The target sites of IS4Bsu1 were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS4Bsu1 in mutant comP genes. These results indicate that IS4Bsu1 transposes by the replicative mechanism, in contrast to other IS4 members that use the conservative mechanism, and that most, if not all, of spontaneous γPGA(−) mutants appear to have resulted from the insertion of IS4Bsu1 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 IS4Bsu1 in B. subtilis (natto).