Takashi Ano

Production of the antifungal peptide antibiotic, iturin by Bacillus subtilis NB22 in solid state fermentation

Abstract Production of iturin, an antifungal peptide effective at suppressing phytopathogens, by Bacillus subtilis NB22 was investigated in solid state fermentation (SSF) using soy bean curd residue ( okara ). In scale-up from 15 g to 3 kg, aeration, temperature, and moisture content were controlling factors for the efficient production of iturin. It was found that solid state fermentation was 6–8 times more efficient with respect to iturin productivity than submerged fermentation on the basis of unit wet weight. Higher productivity in selective production of specific components of iturin which are stronger inhibitors of plant pathogens was also confirmed in SSF.

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8.

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides.

Effect of Temperature on Production of Lipopeptide Antibiotics, Iturin A and Surfactin by a Dual Producer, Bacillus subtilis RB14,in Solid-State Fermentation

Abstract Temperature dependency of the production of iturin A and surfactin by a dual producer, Bacillus subtilis RB14, in the solid-state fermentation of okara was investigated. The optimal temperature for iturin A was 25°C, while that for surfactin was 37°C, in spite of their dependency on a common gene, lpa-14 . When the effect of temperature on the relative ratios of the amount of the five homologues of iturin A to the total iturin A produced by RB14 was investigated, only the ratios of homologues having β-amino acid residues with normal aliphatic chains were affected and the ratio of the homologue with longer normal (C 16 -) chain increased as the temperature was increased.

Use of soybean curd residue, okara, for the solid state substrate in the production of a lipopeptide antibiotic, iturin A, by Bacillus subtilis NB22

Abstract The possibility of the utilization of soybean curd residue, okara, for the production of a lipopeptide antibiotic, iturin A, in solid state fermentation (SSF) by Bacillus subtilis NB22 was investigated. Okara is a by-product of the tofu manufacturing process, now treated as an industrial waste and disposed of mostly by incineration. Dehydrated okara, with improved transportability and preservability, could be used as effectively as the fresh, intact okara for SSF by B. subtilis NB22 for the production of iturin A.

Nucleotide sequence and characteristics of the gene, lpa-14, responsible for biosynthesis of the lipopeptide antibiotics iturin A and surfactin from Bacillus subtilis RB14

Abstract Bacillus subtilis RB14 is a coproducer of the lipopeptide antibiotics iturin A and surfactin. Iturin A and surfactin have a similar structure consisting of a seven-amino-acid cyclic peptide, linked by either hydroxy or ester peptide linkage, respectively, to the fatty acid part. A 10-kb fragment responsible for the production of the lipopeptide antibiotics iturin A and surfactin in B. subtilis RB14 was minimized and the nucleotide sequence of the region essential for the synthesis of the lipopeptides was determined. A large open reading frame consisting of 224 amino acid residues was found. The gene, designated as lpa-14 (lipopeptide antibiotic production of RB14), showed high homology with sfp and psf-1 (regulators for surfactin production in B. subtilis and Bacillus pumilus, respectively) and an unknown open reading frame, orfX, in the upstream region of the peptide antibiotic gramicidin S biosynthesis operon of Bacillus brevis. The biosynthesis of surfactin and of iturin A was shown to be coregulated by the same gene, lpa-14. It is suggested that a common regulation system might exist among these genes for the production of peptide antibiotics in Bacillus species.

Production of antifungal antibiotic, iturin in a solid state fermentation byBacillus subtilis NB22 using wheat bran as a substrate

SummaryProduction of a family of lipopeptide antibiotic, iturin byB. subtilis NB22, in solid state fermentation (SSF) of wheat bran (WB) was investigated. The amount of iturin produced per unit weight of wet substrate was 5–6 times more than that in the submerged fermentation (SMF). SSF enabled to produce a homologue of iturin with strong antibiotic activity in a larger fraction compared with the SMF.

Horizontal Transfer of Iturin A Operon, itu, to Bacillus subtilis 168 and Conversion into an Iturin A Producer

ABSTRACT Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three iturin group operons (i.e., iturin A, mycosubtilin, and bacillomycin D) of those antibiotic-producing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb iturin A operon, was transferred via competent cell transformation to the genome of a non-iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The iturin A operon-transferred strain 168 was then converted into an iturin A producer by the introduction of an sfp gene, which encodes 4′-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.

Effect of homogeneous and inhomogeneous high magnetic fields on the growth of Escherichia coli

Abstract The growth of Escherichia coli B was investigated under homogenous 7 Tesla (T) magnetic field, and inhomogeneous 5.2∼6.1 T and 3.2∼6.7 T magnetic fields which are produced by a newly constructed superconducting magnet biosystem (SBS). These high magnetic field adversely affected on the growth of the bacterium in the early logarithmic growth phase. However, in the stationary phase, the cell number under a high magnetic field was about 2∼3 times higher than that of a control, indicating that the magnitude of the decrease in the cell number was reduced by the high magnetic field. The effect of the inhomogeneous magnetic field was much stronger than that of the homogeneous one. The high magnetic field was found to affect the cells of the bacterium differently, depending on the growth phases.

Effect of super high magnetic field on the growth of Escherichia coli under various medium compositions and temperatures

Abstract When two strains of Escherichia coli were grown under a super high magnetic field (11.7 Tesla) in complex medium, the growth was stimulated in comparison with that in the geomagnetic field. When the bacteria were grown in synthetic medium, the growth rates were reduced significantly. As a result of the addition of casamino acids to the synthetic medium, the growth was shifted from a reduced state to a stimulated one, suggesting that certain amino acids are responsible for the phenomenon. Twenty amino acids were thus added individually to minimal medium; some of these amino acids shifted the reduced growth state to accelerated or normal growth. The critical concentration of glutamic acid for the growth shift was determined to be 0.01–0.1 mg/l. When the bacteria were grown at temperatures lower than the optimal temperature for growth, the 11.7 T magnetic field enhanced the growth rate irrespective of media used, while at higher temperatures reduced growth became significant in accordance with the increase in temperature. A potential use of the high magnetic field as a control factor in biological system is suggested.

High magnetic field enhances stationary phase-specific transcription activity of Escherichia coli.

When Escherichia coli B was aerobically grown at 37 degrees C under inhomogeneous 5.2-6.1 Tesla (T) magnetic fields in the superconducting magnet biosystem (SBS), the cell number in the stationary phase after the growth had leveled off, was about 3 times higher than that under a geomagnetic field. When the E. coli defective in the rpoS gene which encodes a sigma factor, sigmaS of RNA polymerase and is specifically expressed in the stationary phase was cultivated at 37 degrees C in SBS, such enhancement of cell survival was significantly reduced. The E. coli cells carrying rpoS-lacZ fusion gene or other rpoS dependent genes fused with lacZ were grown, significant increase in the activity of beta-galactosidase was observed in the stationary phase under high magnetic field. These data suggest that enhancement of the transcription activity in stationary phase is involved in the higher survival of the cells under magnetic field.