T. Nagai et al., A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-glutamic acid production in Bacillus subtilis, J BACT, 182(9), 2000, pp. 2387-2392
Certain Bacillus subtilis strains, such as B. subtilis (natto) starter stra
ins for the manufacture of natto (fermented soybeans), produce capsular pol
y-gamma-glutamate (gamma PGA). In B. subtilis (natto), gamma PGA 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 ne
w insertion sequence (IS), designated IS4Bsu1, in the comP gene of a sponta
neous gamma PGA-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 simil
arity to the Tpases of IS4 family members. Southern blot analyses have iden
tified 6 to 11 copies of IS4Bsu1, among which 6 copies mere at the same loc
i, in the chromosomes of B. subtilis (natto) strains, including NAF4, three
commercial starters, and another three gamma PGA-producing B. subtilis (na
tto) strains. All of the eight spontaneous gamma PGA(-) 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 re
gion. The target sites of IS4Bsu1 mere determined to be AT-rich 9-bp sequen
ces by sequencing the flanking regions of IS4Bsu1 in mutant comP genes. The
se results indicate that IS4Bsu1 transposes by the replicative mechanism, i
n contrast to other IS4 members that use the conservative mechanism, and th
at most, if not all, of spontaneous gamma PGA(-) mutants appear to have res
ulted from the insertion of IS4Bsu1 exclusively into comP. The presence of
insertion hot spots in comP, which is essential for gamma PGA synthesis, as
well as high transposition activity, would account for the high frequency
of spontaneous gamma PGA(-) mutation by IS4Bsu1 in B. subtilis (natto).