A new IS4 family insertion sequence, IS4Bsu1, responsible for genetic instability of poly-gamma-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-gamma-glutamate (gammaPGA). In B. subtilis (natto), gammaPGA 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 gammaPGA-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 gammaPGA-producing B. subtilis (natto) strains. All of the eight spontaneous gammaPGA(-) 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 gammaPGA(-) 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 gammaPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous gammaPGA(-) mutation by IS4Bsu1 in B. subtilis (natto).     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange.

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis

In Bacillus subtilis, the ComQXPA quorum-sensing system controls cell density-dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly-gamma-glutamate (gamma-PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917-LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917-LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that gamma-PGA synthesis in B. subtilis (natto) is under the control of the ComP-ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP:cat) and gamma-PGA production of B. subtilis (natto) NAF12 (comP:Tn917-LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild-type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.     

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.     

Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto)

Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.     

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.     

Heterogenous expression of poly-gamma-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3

Bacillus licheniformis WBL-3, one of poly-gamma-glutamic acid (gamma-PGA) producers, depends on the existence of glutamate in the medium. In this paper, gamma-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgsBCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IF03336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced gamma-PGA extracellularly. The yield of gamma-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar gamma-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize gamma-PGA.     

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.     

Structure of the hydrolyzed product (F-2) released from gamma-polyglutamic acid by gamma-glutamyl hydrolase YwtD of Bacillus subtilis

The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from gamma-polyglutamic acid by the gamma-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the gamma-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.     

Natto mucilage containing poly-gamma-glutamic acid increases soluble calcium in the rat small intestine

We prepared natto (fermented soybeans) mucilage containing poly-gamma-glutamic acid (gamma-PGA) from commercial natto. The effect of natto mucilage on calcium (Ca) solubility in vitro and in vivo was investigated. Ca solubility in vitro increased with an increase in the amount of natto mucilage, due to inhibition of the formation of an insoluble complex of Ca with phosphate by natto mucilage. Rats were fed with 5 g of soybean protein isolate, natto, mucilage-free natto, or natto mucilage diet for 1.5 h. Small intestinal contents were collected 2.5 h after ingestion. In the lower half of the small intestine, both the amount and the percentage of soluble Ca of intestinal contents were significantly higher (P < 0.001) in rats fed with natto mucilage diet than in those fed with the other diets. Natto mucilage also increased Ca solubility in vivo. These results suggested that gamma-PGA is responsible for the increasing effect of natto mucilage on Ca solubility.     

Characterization of a Generalized Transducing Phage of Poly-(gamma)-Glutamic Acid-Producing Bacillus subtilis and Its Application for Analysis of Tn917-LTV1 Insertional Mutants Defective in Poly-(gamma)-Glutamic Acid Production

A generalized transducing phage, (phi)BN100, was isolated from a Bacillus subtilis (natto) strain producing poly-(gamma)-glutamic acid ((gamma)PGA). Transduction frequencies for a given marker ranged from 3.8 x 10(sup-8) to 1.6 x 10(sup-6) per phage particle. The genome size of the phage was approximately 42 kb. (phi)BN100 was used successfully to identify bona fide Tn917-LTV1 transpositional mutants defective in (gamma)PGA production.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Enhanced glutamic acid production by a H+-ATPase-defective mutant of Corynebacterium glutamicum

Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 microg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 microg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 microg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.     

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.     

Characterization of the Bacillus subtilis ywtD gene,whose product is involved in gamma-polyglutamic acid degradation

The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.     

Simultaneous presence of three cryptic plasmids of various size in a soil strain of Bacillus subtilis]

The Bacillus subtilis 1387 soil strain, which contains three cryptic plasmids simultaneously, was described. Two small plasmids (6.3 and 8.5 kb) were homologous to each other, and a large plasmid (30 kb) had no homology with them. The plasmids were separately transmitted into cells of the Bac. subtilis 168 strain, and some plasmid characteristics were analyzed.     

Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance

To invent a functional natto promoting bone formation, the construction of a strain with high productivity of vitamin K2 (menaquinone-7: MK-7), which is important in the carboxylation of a kind of bone protein participating in bone formation, osteocalcin, was investigated. To screen for a strain appropriate to making natto (a Japanese traditional fermented soybean food) with high productivity of MK-7, a combination of analog resistance to the compounds on the biosynthetic pathway of menaquinones with mutation was done. Consequently, strain OUV23481, with 2-fold higher productivity (1,719 microg/100 g natto) of MK-7 than that of a commercial strain, was constructed as a mutant with analog resistance to 1-hydroxy-2-naphthoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), m-fluoro-D,L-phenylalanine (mFP), and beta-2-thienylalanine (betaTA). This strain was classified as Bacillus subtilis (natto). The natto made using this strain was evaluated to have a good quality as natto in all the viewpoints of appearance, flavor, taste, texture, and stringiness.     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.