Subtilis 168 Research Articles

Improvement of Fibrinolytic Activity of Bacillus subtilis 168 by Integration of a Fibrinolytic Gene into the Chromosome.

Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy(-), Spec(S)) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.

Genes of Bacillus subtilis 168 that Support Growth of the Cyanobacterium, Synechococcus leopoliensis CCAP1405/1 on Agar Media.

Synechococcus leopoliensis CCAP1405/1 cannot grow on common solid media; however, the strain can grow when co-cultured with Bacillus subtilis 168. Gene-disruptant strains of B. subtilis 168 by pMUTINs at the following sites lost the ability to support the growth of S. leopoliensis CCAP1405/1 on agar media: yxeO and yufO (transporter), yxdK (histidine kinase), sdhC (succinate dehydrogenase), yvgQR (sulfite reductase), acoB (acetoin dehydrogenase), yusE (thioredoxin), yrdA (function unknown). Involvement of the assimilatory sulfate reduction pathway was the suggested reason for loss of the function.

Enhanced activity of an alkaline phytase from Bacillus subtilis 168 in acidic and neutral environments by directed evolution

As a feed additive, the desirable features of phytase include thermostability, strict substrate specificity, and high stability in a wide pH range. Alkaline phytases from Bacillus subtilis are promising candidates because of their high inherent thermostability and substrate specificity, but the application is restricted due to their poor specific activity in acidic and neutral conditions. Directed evolution was utilized in this paper to improve the activity of phytase from B. subtilis 168 in neutral and acidic environments. After two rounds of evolution, a number of positive mutants were picked out from the mutant library. Compared with the wild type, variants D24G, S51A, D24G/K265N, D24G/K70R/K111E/N121S showed 29.7%, 13.5%, 42.7%, 42.8% improvement in specific activity under the optimal reaction conditions (pH 7.0, 60°C) and 76.6%, 79.5%, 84.2%, 121.1% improvement at pH 4.5 and 37°C, respectively. The catalytic efficiency (kcat/Km) of D24G, D24G/K265N, D24G/K70R/K111E/N121S at pH 7.0 was increased by 132%, 110%, 131% as compared with that of the wild type, and the improvement at pH 4.5 was 98.0%, 114.5%, 163.3%, respectively. Molecular docking results revealed that D24, S51 and K70 had a relatively large influence on the catalytic activity probably because they are adjacent to the active site cleft.

Improved Production of Sublancin 168 Biosynthesized by Bacillus subtilis 168 Using Chemometric Methodology and Statistical Experimental Designs.

Sublancin 168, as a distinct S-linked antimicrobial glycopeptide produced by Bacillus subtilis 168, is effective in killing specific microorganisms. However, the reported yield of sublancin 168 is at a low level of no more than 60 mg from 1 L fermentation culture of B. subtilis 168 by using the method in the literature. Thus optimization of fermentation condition for efficiently producing sublancin 168 is required. Here, Box-Behnken design was used to determine the optimal combination of three fermentation parameters, namely, corn powder, soybean meal, and temperature that were identified previously by Plackett-Burman design and the steepest ascent experiment. Subsequently, based on the response surface methodology, the quadratic regression model for optimally producing sublancin 168 was developed, and the optimal combination of culture parameters for maximum sublancin 168 production of 129.72 mg/L was determined as corn powder 28.49 g/L, soybean meal 22.99 g/L, and incubation temperature 30.8°C. The results showed that sublancin 168 production obtained experimentally was coincident with predicted value of 125.88 mg/L, and the developed model was proved to be adequate, and the aim of efficiently producing sublancin 168 was achieved.

Sustainable endospore-based microreactor system for antioxidant capacity assay.

A novel endospore-based microbial method for "post-additional" antioxidant capacity assay was developed. The technique was based on oxidation and catalysis of the 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) by Bacillus subtilis 168 endospores in the presence of dissolved oxygen. Coat protein A (CotA), which belongs to the endospore coat, was expressed, purified, and assessed for its ability to oxidize ABTS into the ABTS(•+) radical cation. The wild-type endospore necessary for oxidizing ABTS into ABTS(•+) radical cation was confirmed by knocking out the cotA gene from B. subtilis 168 by homologous double exchange. Findings revealed that the catalytic activity of the endospores may be attributed to the presence of the CotA protein. The use of endospores instead of purified enzymes to prepare ABTS(•+) greatly reduced the assay cost and eliminated the need to purify and store of enzymes. The self-life of the radical cation was kept stable for at least 12 days without addition of a stabilizer and laccase inhibitor. This behavior enables the large-scale preparation of ABTS(•+). The antioxidant capacities of the individual antioxidants and fruit samples were easily quantified and compared using the proposed method. The developed technique can be further developed as a high-throughput screening technique for antioxidants.

Application of Bacillus subtilis 168 as a Multifunctional Agent for Improvement of the Durability of Cement Mortar

Microbiological calcium carbonate precipitation (MCCP) has been investigated for its ability to improve the durability of cement mortar. However, very few strains have been applied to crack remediation and strengthening of cementitious materials. In this study, we report the biodeposition of Bacillus subtilis 168 and its ability to enhance the durability of cement material. B. subtilis 168 was applied to the surface of cement specimens. The results showed a new layer of deposited organic-inorganic composites on the surface of the cement paste. In addition, the water permeability of the cement paste treated with B. subtilis 168 was lower than that of non-treated specimens. Furthermore, artificial cracks in the cement paste were completely remediated by the biodeposition of B. subtilis 168. The compressive strength of cement mortar treated with B. subtilis 168 increased by about 19.5% when compared with samples completed with only B4 medium. Taken together, these findings suggest that the biodeposition of B. subtilis 168 could be used as a sealing and coating agent to improve the strength and water resistance of concrete. This is the first paper to report the application of Bacillus subtilis 168 for its ability to improve the durability of cement mortar through calcium carbonate precipitation.

Characterization and optimization of Bacillus subtilis ATCC 6051 as an expression host

The genome sequence of Bacillus subtilis ATCC 6051 and its suitability as an expression host for recombinant protein production was determined. The comparison of this undomesticated wild type with the widely used laboratory strain B. subtilis 168 reveals a high degree of congruency between the two strains. Differences could only be detected on the level of point mutations or small insertions. B. subtilis ATCC 6051 shows none of the auxotrophies known for B. subtilis 168 and is able to produce polyketides. It exhibits better use of complex media and higher genomic stability through reduced natural competence. Consequently, B. subtilis ATCC 6051 was genetically modified to yield an optimized strain for the production of heterologously expressed proteins under control of an acetoin-inducible promoter.

Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data

BackgroundBacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.ResultsWe applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1.These are specific for γ-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.ConclusionsThe determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http://natto-genome.org/.

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein γ-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80.

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.

Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368.

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15 beta position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15 beta-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.

Relation between wall teichoic acid content and Concanavalin A binding in Bacillus subtilis 168

It has been known for some time that Concanavalin A will agglutinate bacteria whose wall teichoic acids contain a-linked glucosyl substituents [1]. The ability of the lectin to precipitate appropriately substituted teichoic acids has been used in structural [2] and preparative [3] studies and its interaction with whole bacteria or isolated walls has been studied in attempts to gain information on the location of teichoic acids in the cell wall and at the bacterial surface [4,5]. Interpretation of the latter studies has been complicated by the lack of any information on how the amount of Concanavalin A bound is related to the amount of teichoic acid present in the bacteria, or whether variations in the amount of teichoic acid present affect both the amount bound and the affinity of binding. We have recently [6] shown that the amount of teichoic acid present in walls of B. subtilis 168 can be controlled by growing the bacteria under balanced conditions with varied phosphate supply. We now report a study of the interaction of these bacteria with Concanavalin A. Our results show that the association constant of the binding reaction is little affected by variation in teichoic acid content but the amount of Concanavalin A bound increases in proportion to the amount of teichoic acid present although it ap-

Isolation and characterization of a bacteriophage forBacillus licheniformis A5

The isolation of a virulent bacteriophage forBacillus licheniformis A5 is reported. This bacteriophage, designated NLP-1, has an icosahedral head 100 nm in diameter and a contractible tail with a maximum length of 130 nm. Its DNA has a density of 1.741 g/cm3 and aT m of 78.4°C. Base composition analysis showed that thymine is absent and is replaced by hydroxymethyluracil. NLP-1 appears to belong to theBacillus group of bacteriophages that includes SP01 and SP82. It will infectB. cereus T andB. brevis 8185, but will not infectB. subtilis W23 or 168.

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis.

We constructed transformation of B. subtilis 168 which acquired genes for site-specific restriction endonucleases. These endonucleases originated from various strains of B. subtilis and were classified into five groups based on the specificity of the sequences recognized by the enzymes. We examined the loci of genes for site-specific restriction endonucleases belonging to different groups: hsrE determined Endo. R. Bsu1231 (I), hsrB Endo.R.Bsu1247(I), hsrR Endo.R.BsuR and hsrC Endo.R.Bsu-1247(II). One gene, hsrE, was located between sacA and purA by transduction crosses with phage PBS1, and another gene, hsrB, between hsrE and purA. Genes hsrR and hsrC had been suggested to be allelic or closely linked by previous studies with transformation. We located hsrR and hsrC between purB and tre. Our previous observation and this study show that B. subtilis 168 has at least three independent loci on the chromosome for four genes for site-specific restriction endonucleases in addition to the locus for the original restriction activity (Bsu168-specific restriction) of strain 168.

Plasmid deoxyribonucleic acid in Bacillus subtilis and Bacillus pumilus.

Two of eighteen strains of Bacillus subtilis examined contained covalently closed circular duplex deoxyribonucleic acid (DNA) of homogeneous size and buoyant density. Strain ATCC 15841 contained about 16 copies per chromosome of plasmid pPL1, a circular DNA element having a molecular weight of about 4.7 times 10(6) and a buoyant density of 1.700. Strain ATCC 7003 contained about one to two copies per chromosome of plasmid pPL2. pPL2 had a molecular weight of about 46 times 10(6) and a buoyant density of 1.696. Strain ATCC 7003 appeared to be closely related to B. subtilis 168 by genetic, physiological, and biochemical criteria. Strain ATCC 15841 appeared to be much less closely related. B. pumilus ATCC 12140 contained two size classes of covalently closed circular duplex DNA. The plasmids pMB1 and pMB2 had molecular weights of about 6.8 times 10(6) and 5.3 times 10(6), respectively, and were present in several copies per chromosome.