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

Abstract

Certain Bacillus subtilis strains, such as B. subtilis (natto) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-␥-glutamate (␥PGA). In B. subtilis (natto), ␥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 new insertion sequence (IS), designated IS4Bsu1, in the comP gene of a spontaneous ␥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 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 ␥PGA-producing B. subtilis (natto) strains. All of the eight spontaneous ␥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 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 ␥PGA ؊ 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 ␥PGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous ␥PGA ؊ mutation by IS4Bsu1 in B. subtilis (natto).

Bacteriophages of Bacillus subtilis (natto) and Their Contamination in Natto Factories

“…The gene pgsBCA is homologous with genes for capsular PGA production of B. anthracis and codes for a membranous enzyme complex (Ashiuchi et al, 2001). On the other hand, a regulatory gene for PGA production was cloned and found to be comP, which codes for a sensor protein kinase of the ComP-ComA two-component signal transduction system (Nagai et al, 2000). Recently, PGA cross-linked by γ-radiation was found to hold a large quantity of water and to be useful for greening of desert areas by scattering the PGA resin in which seeds (e.g.…”

Characterization of <i>Bacillus subtilis </i>Strains Isolated from Fermented Soybean Foods in Southeast Asia: Comparison with <i>B</i>. <i>subtilis </i>(<i>natto</i>) Starter Strains

“…DNA fragments were Southern-blotted using the 1.2-kb BamHI-PstI fragment of IS4BSu1 as a probe 12 . To determine the IS4Bsu1sequences, the tnpA region from nucleotide positions (nt) 124 to 1301 of the IS 12 was amplified from the chromosomal DNAs of B. subtilis strains using the oligonucleotide primers, 5'-AAGGACAATAAGCATGGATAAG-3' (nt 124 to 145) and 5'-ACTATAATCTTTGACGAGTGCA-3' (complementary to nt 1280 to 1301), and KOD DNA polymerase (Toyobo Biochemicals). Amplified 1.2-kb DNA fragments were then purified by agarose gel electrophoresis and used as sequencing templates along with the above primer pair, the following primers, 5'-GTTCATAATC-CAAGTAACCCG-3' (nt 746 to 725), 5'-AGGTGCT-GCTTCAGCCATTTAA-3' (complementary nt 1032 to 1053) and a Dye-Terminator Cycle Sequencing Kit (Perkin-Elmer).…”

“…Restriction endonuclease digestion, agarose gel electrophoresis, and polymerase chain reaction (PCR) were performed as described 12 . DNA fragments were Southern-blotted using the 1.2-kb BamHI-PstI fragment of IS4BSu1 as a probe 12 .…”

“…Restriction endonuclease digestion, agarose gel electrophoresis, and polymerase chain reaction (PCR) were performed as described 12 . DNA fragments were Southern-blotted using the 1.2-kb BamHI-PstI fragment of IS4BSu1 as a probe 12 . To determine the IS4Bsu1sequences, the tnpA region from nucleotide positions (nt) 124 to 1301 of the IS 12 was amplified from the chromosomal DNAs of B. subtilis strains using the oligonucleotide primers, 5'-AAGGACAATAAGCATGGATAAG-3' (nt 124 to 145) and 5'-ACTATAATCTTTGACGAGTGCA-3' (complementary to nt 1280 to 1301), and KOD DNA polymerase (Toyobo Biochemicals).…”

“…Production of γ-PGA in these strains is regulated by the ComQXPA quorum sensing system 7,19,20 , and is genetically unstable. This genetic instability is caused by the translocation of the insertion sequence (IS) IS4Bsu1 on the chromosome into the comP gene at a high frequency 11,12 . The comP gene encodes a bifunctional protein, having both sensor (for ComX pheromone) and transmitter functions that transfer the pheromone-signal to the ComA response regulator 19 .…”