Lack of Nucleotide Sequence Relationship Among the Temperate Bacteriophage SPO2, Bacillus subtilis, and Virulent Bacteriophages β3 and β22

Radiolabeled phage SPO2 fragments were tested for the abiity to form interspecies deoxyribonucleic acid (DNA) duplexes with DNA from Bacillus subtilis and from phages beta3 and beta22. No reassociation, above control values, occurred between the DNA of phage SPO2 and that of its host or either of the virulent B. subtilis phages. It appears that extensive nucleotide sequence similarities between portions of host and phage DNA species are not necessary for the formation of the lysogenic state. The thermal elution profile of reassociated SPO2 DNA exhibited a normal distribution, indicating no heterogeneity in base composition. This differs from temperate enterophages whose DNA molecules contain regions of differing base composition.     

Bacillus subtilis Bacteriophage SP01, SP82, and φe Require Host Lysyl- and Tryptophanyl-tRNA Synthetases for Phage Development

Infection of Bacillus subtilis mutants having temperature-sensitive lysyl- or tryptophanyl-tRNA synthetases with bacteriophage SP01, SP82, or phie indicates that both host enzymes are essential for phage development. No apparent modification of the temperature-sensitive phenotype of the mutant host enzymes occurs during phage infection.     

Restriction fragment maps of the genome of Bacillus subtilis bacteriophage SPβ

Six different restriction endonucleases were used to generate restriction fragment maps of the genome of the temperate Bacillus subtilis phage SPβ. AvaI and SalI each had six target sites in the phage DNA, AvaII had three, BamHI had seven, PstI had twenty, and SacI had sixteen. Restriction analysis and heteroduplex analysis were used to locate a 10-kb region of DNA that is deleted in the clear-plaque mutant, spβci. Thedeletion lay approx. 50 kb from the left end of the 126-kb phage genome.     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

Occurrence of Two Types of Cystathionine β-Cleavage Enzyme in Bacillus subtilis

Production of extracellular polysaccharidases by Irpex lacteus Fr. was studied in different culture conditions.

The presence of fatty acids such as linolic, linolenic, erucic, palmitic acids etc. caused remarkably to increase the production of ceîlulase (filter paper disintegrating activity, FD), laminarinase and xylanase. On the contrary, fatty acids had not any special effect on the production of cellulolytic enzymes such as Avicelase and CMCase and of plant tissue macerating enzymes (MA).

When two kinds of carbon sources, e.g., cellulose powder and potato pulp were mixed together and used as an inducer, polysaccharidase production investigated, with the exception of CMCase, increased higher than when the two substances were used separately.     

Relation between wall teichoic acid content of Bacillus subtilis and efficiency of adsorption of bacteriophages SP 50 and Φ 25

Efficient adsorption of bacteriophages SP 50 and 25 occurred only to bacilli that contained wall teichoic acid and neither phage bound to phosphate limited bacilli that contained teichuronic acid instead of teichoic acid. Though both phages require the presence of teichoic acid, their receptors are not identical. Efficient binding of phage 25 required the presence of greater proportions of teichoic acid in the wall and the receptor for this phage was destroyed when bacteria or isolated walls were heated at pH 4 whereas the ability of these samples to bind phage SP 50 was unaffected by such treatment. Efficient binding of phage SP 50 was not highly dependant on the presence of glucosyl substituents on the teichoic acid. Such substituents were required for phage 25 binding though their anomeric configuration appeared to be unimportant since the phages bound well to both strains W23 and 168, the wall teichoic acids of which carry glucosyl substituents of opposite anomeric configuration. The differeneces in the nature of the receptors may be of value in the use of the phages as probes for the location and distribution of teichoic acid in the wall.Non-Standard Abbreviation PAE Phage adsorption efficiency, as defined by Archibald and Coapes (1976)     

Purification and enzymatic characterization of PgcM: a β-phosphoglucomutase and glucose-1-phosphate phosphodismutase of Bacillus subtilis

Phosphoglucomutases catalyze the reversible conversion of D-glucose 1-phosphate to D-glucose 6-phosphate, a key metabolic step in all cells. Two classes of phosphoglucomutases have been described so far, using either the alpha- or beta-forms of the phosphorylated sugars. The pgcM gene of Bacillus subtilis was cloned and used to construct a plasmid-based overexpression system for PgcM in Bacillus megaterium. The obtained protein was purified and its enzymatic activities were characterized. PgcM exhibits beta-phosphoglucomutase activity, transforming mainly beta-glucose 1-phosphate to beta-glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Nevertheless, alpha-glucose 1-phosphate can also serve as a substrate, but with a seven-fold lower affinity than that observed for the beta-form. Additionally, PgcM exhibits a glucose-1-phosphate phosphodismutase activity using the alpha- and beta-forms as substrates, with affinities comparable to those observed for the phosphoglucomutase activity. Conformational changes of PgcM triggered by cofactors (MgCl2, glucose 1,6-bisphosphate) and substrate (glucose 1-phosphate) were detected by fluorescence spectra. Insertional mutagenesis of pgcM resulted in an inactivation of beta-phosphoglucomutase activity in B. subtilis. These mutants showed growth deficiency on minimal medium containing starch or maltodextrins (maltose to maltoheptaose) compared either to the wild-type or to growth on minimal medium containing glucose.     

Cloning and expression of β-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Summary The -galactosidase gene from Bacillus subtilis KL88 was cloned into Escherichia coli and the gene product characterized for its potential use in the dairy industry. The two recombinant plasmids that we obtanied encoded a -galactosidase with the same catalytic and thermal characteristics as the native -galactosidase from B. subtilis. The recombinant -galactosidases exhibited high activity at low temperature (10°C), with maximum activity at 50°C and an optimum pH of 6.0. Its molecular weight was estimated to be 90 Kd. The restriction maps of the recombinant plasmids were constructed. The -galactosidase gene was located in a 2.3 Kb fragment.     

Recombinant β-glucanase production and plasmid stability of Bacillus subtilis in cyclic fed batch culture

Recombinant -glucanase production and plasmid stability were higher in cyclic fed batch culture than in batch and chemostat culture. Plasmid stability was maintained indefinitely in cyclic fed batch cultures of Bacillus subtilis when subjected to cycle periods of 2 or 4 hours. Cycle periods greater than 6 hours resulted in loss of the plasmid. In batch and chemostat cultures plasmid loss was rapid. -1,3-glucanase productivity was highest in cyclic fed batch culture.     

Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

Propeptide of Bacillus subtilis amylase enhances extracellular production of human interferon-α in Bacillus subtilis

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 × 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

α-Amylase Formation and Nucleic Acid Metabolism of Bacillus subtilis

The nucleic acid metabolism in washed cells of Bacillus subtilis was investigated with special reference to amylase formation of the bacterium. On incubation of the suspension of the washed cells, purines, pyrimidines and their related compounds were observed in the medium. However, in the medium of the cells incubated with a calcium chelater, where no amylase formation occurred, were detected adenosine- and guanosine-monophosphate in addition to those described above. The addition of a calcium chelater was also found to decrease the quantity of the nucleic acids being involved in the lysozyme-sensitive fraction of the bacterial cells, suggesting the possibility that the metabolism of nucleic acids in this fraction is closely related to amylase formation of the cells.     

Parametric analysis of metabolic fluxes of α-amylase and protease-producing Bacillus subtilis

Parametric analysis was applied for a metabolic flux model for the fed-batch culture of Bacillus subtilis producing recombinant α-amylase and protease. The metabolic flux model was formulated as a linear programming problem consisting of 49 reactions (decision variables) and 50 metabolites (equality constraints). This study was aimed to determine the response of the metabolic fluxes and objective function value of minimizing the difference between ATP consumption and ATP production (ATP balance). With regard to intracellular metabolite accumulation, the objective function value was least sensitive to variation in succinate and most sensitive to variation in malate. Amongst the variations in the accumulation rates of extracellular metabolites, the objective function value was least sensitive to variation in glutamate and most sensitive to variation in starch hydrolysis and triglyceride synthesis. A 10% variation in metabolite accumulation rates caused a maximum of 13.8% variation (standard error = 3.8%) in the objective function value.     

Cloning and Nucleotide Sequence of the Bacillus subtilis K trp B Gene Encoding Tryptophan Synthase β-Subunit

When defatted soy milk was ultracentrifuged, 34kDa allergenic soybean protein Gly m Bd 30 К was more abundant in the precipitate than in the supernatant by an SDS-PAGE analysis. The addition of more than 10 тм of 2-mercaptoethanol (2-ME) to the soy milk resulted not only in further removal of the 34 kDa allergenic protein to the precipitate, but also in better recovery of conglycinin in the supernatant.

After a two-dimensional SDS-PAGE analysis (the first dimension, minus 2-ME; the second, plus 2-ME) of the precipitates, superimposition between the CBB-stained gel and the electroblotted membrane stained with a monoclonal antibody specific to Gly m Bd 30 К indicated that part of Gly m Bd 30 К was preferentially bound to the a’- and a-subunits of conglycinin, and that part of them had formed the dimer through a disulfide bond.