Control of ��-amylase production by Bacillus subtilis

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.     

Secretion of biologically-active human interferon-β by Bacillus subtilis

Human interferon-β (hIFN-β) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-β with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-β with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-β in B. subtilis.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

α-Amylase production by immobilized whole cells of Bacillus subtilis

Summary Whole cells of Bacillus subtilis were immobilized in polyacrylamide gel prepared from 5% total acrylamide (85% acrylamide and 15% N,N-methylenebisacrylamide). Production of -amylase by the immobilized whole cells was attempted in a batch system. -Amylase produced by the immobilized whole cells was about three times larger than that produced by washed cells at optimum conditions. The reusability of the immobilized whole cells and washed cells was examined. The activity of -amylase production by washed cells decreased with increasing use cycles. On the other hand, the activity of the immobilized cells increased gradually, and it reched a steady state after seven cycles. -Amylase was produced from a simple reaction medium containing 1% meat extract and 0.05% yeast extract by the immobilized whole cells. The rate of -amylase production by the immobilized whole cells was the same as in submerged cultivation using starch bouillon medium. Growth of B. subtilis in polyacrylamide gel was observed by electron microscopy.     

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.     

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.     

Abortive Infection of Sporulating Bacillus subtilis 168 by φ2 Bacteriophage

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

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.     

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.The cell envelope of the gram-positive bacterium Bacillus subtilis consists of a single (cytoplasmic) membrane surrounded by a relatively thick cell wall consisting of similar proportions of peptidoglycan and covalently attached anionic polymers. The absence of an outer membrane means that there is no equivalent of the membrane-enclosed periplasm found in gram-negative bacteria. However, by virtue of its thickness and high density of negative charge, the cell wall may perform some of the roles of the periplasm in gram-positive bacteria.The absence of an outer membrane in gram-positive bacteria also simplifies the secretion pathway, and, consequently, B. subtilis and its close relatives have the potential to secrete proteins directly into the growth medium, at concentrations in excess of 5 grams per liter (4). Despite its extensive use in the production of commercially important Bacillus enzymes (e.g., α-amylases and alkaline proteases), attempts to exploit B. subtilis for the production of heterologous proteins at high concentrations have proved disappointing (8). One reason for this failure is the production and release into the culture medium of several extracellular proteases (24, 28, 37). Although native Bacillus proteins are generally resistant to these proteases, heterologous proteins are often rapidly degraded in their presence. As a result, strains of B. subtilis that are multiply deficient in extracellular proteases have been developed (11, 37). The more developed of these strains have less than 1% of the proteolytic activity of the wild type (37). To date, efforts have concentrated mainly on the proteases which reside in a truly extracellular location, while those which remain cell associated have been largely overlooked.Although strains deficient in extracellular proteases have improved the productivity of B. subtilis for the production of heterologous proteins, they have only partially overcome problems of unexpectedly low yields. We and others have recently shown (22, 31) that significant amounts of secretory protein are degraded within minutes of being synthesized. This degradation is observed even for Bacillus proteins that are highly resistant to proteases released into the culture medium, suggesting that a component of this degradation is cell associated.Margot and Karamata recently reported the identification of a cell-wall-associated protease encoded by the wprA gene (21). The primary product of this gene is a 96-kDa polypeptide that is processed into two previously identified cell wall proteins, namely, CWBP52 and CWBP23. The processing of the WprA precursor during secretion accompanies the targeting of CWBP52 and CWBP23 to the cell wall and is analagous to the processing of another B. subtilis cell-wall-bound protein, namely, WapA (5). The amino acid sequence of CWBP52 shows a high degree of similarity with serine proteases of the subtilisin family, and phenylmethylsulfonile fluoride (PMSF)-sensitive protease activity was detected in proteins extracted from the cell wall of a wprA⁺ strain, but not one in which this gene had been insertionally inactivated (21). In the absence of homology to proteins in the databases, the N-terminal CWBP23 moiety was presumed to function as a chaperone-like propeptide that is proteolytically processed on the trans side of the membrane. In this paper, we report on a potential role of products of wprA in the integrity of secretory proteins during late stages in the secretion pathway. We also discuss the potential of wprA mutants to increase the productivity of B. subtilis for secretory proteins.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Isolation and Properties of Bacillus subtilis Strains Lysogenized by a Clear Plaque Mutant of Bacteriophage φ105

A clear plaque mutant of the temperate Bacillus phage phi105 lysogenized a small fraction of infected cells forming an integrated prophage at or near the normal phi105 insertion site. These lysogens exhibited a spontaneous induction rate approximately 1,000-fold lower than wild type and were noninducible (ind(-)) by mitomycin C. Prophage was induced, however, when competent cultures were incubated with transforming DNA. The ind(-) phenotype could not be attributed solely to the clear plaque mutation and appears to involve a cell-specific factor. Lysogenization by the clear plaque mutant, in contrast to wild-type phage, did not cause a marked reduction in transformation efficiency.     

Regulation of pro-σK activation: a key checkpoint in Bacillus subtilis sporulation

The Gram-positive bacterium Bacillus subtilis initiates the sporulation process under conditions of nutrient limitation. Here, we review related work in this field, focusing on the protein processing of the pro-σ^K activation. The purpose of this review is to illustrate the mechanism of pro-σ^K activation and provide structural insights into the regulation of spore production. Sporulation is not only important in basic science but also provides mechanistic insight for bacterial control in applications in, e.g., food industry.     

Glucosyl Glycerophosphoric Acid and its Polymer Excreted by α-Amylase-forming Bacillus subtilis

α-Amylase formation by washed cell suspensions of Bac. subtilis was found to be accompanied by the excretion of a compound consisting of glucose, glycerol and phosphoric acid. It was excreted as a polymer and a monomer. The former, a kind of teichoic acid, was significantly dominant in quantity when the cells were incubated under the conditions suitable for α-amylase formation. On the other hand, the monomer prevailed when the bacterial cells were under the unfavorable conditions for the enzyme formation.

Both compounds were purified by ion exchange column chromatography. Chemical and enzymatic investigations revealed the following structures: 2-O-α-d-glucopyranosyl-glycerol-3-monophosphoric acid for the monomer, and a polymerized form of the monomer through phosphodiester linkages involving the hydroxyl groups on C3 of the glycerol, for the polymer.     

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.     

Heat Induction of Prophage φ 105 in Bacillus subtilis: Bacteriophage-Induced Bidirectional Replication of the Bacterial Chromosome

A mutant of Bacillus subtilis, dna-1, which cannot initiate new rounds of DNA replication (obtained from N. Sueoka) was lysogenized with wild-type phi 105 and with the heat-inducible mutant phi 105 cts23. Bacteria were incubated at the permissive temperature in the presence of chloramphenicol and then shifted to the nonpermissive temperature where induction of phi 105 cts23 occurs. DNA made after the shift was labeled with a density label, and the distribution of bacterial and phage markers in replicated and unreplicated DNA was determined. Similar experiments were performed with nonlysogenic dna-1 infected with phage phi 105 cts23 after the temperature shift. The results show that after induction of phi 105 cts23 prophage, bacterial markers on either side of the prophage replicate at an increased rate compared to more distant markers. No selective stimulation of bacterial DNA synthesis was observed on infection or after shifting bacteria lysogenic for noninducible phage to the higher temperature. Attempts to suppress the initiation mutation dna-1 by phage phi 105 were unsuccessful.     

Characterization of Bacillus subtilis uracil-DNA glycosylase and its inhibition by phage φ29 protein p56

Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.