Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

The gene coding for alpha-amylase from Bacillus amyloliquefaciens was isolated by direct shotgun cloning using B. subtilis as a host. The genome of B. amyloliquefaciens was partially digested with the restriction endonuclease MboI and 2- to 5-kb fragments were isolated and joined to plasmid pUB110. Competent B. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. One of the transformants producing high amounts of alpha-amylase was characterized further. The alpha-amylase gene was shown to be present in a 2.3-kb insert. The alpha-amylase production of the transformed B. subtilis could be prevented by inserting lambda DNA fragments into unique sites of EcoRI, HindIII and KpnI in the insert. Foreign DNA inserted into a unique ClaI site failed to affect the alpha-amylase production. The amount of alpha-amylase activity produced by this transformed B. subtilis was about 2500-fold higher than that for the wild-type B. subtilis Marburg strain, and about 5 times higher than the activity produced by the donor B. amyloliquefaciens strain. Virtually all of the alpha-amylase was secreted into the culture medium. The secreted alpha-amylase was shown to be indistinguishable from that of B. amyloliquefaciens as based on immunological and biochemical criteria.     

Genetic Control of the Rate of α-Amylase Synthesis in Bacillus subtilis

The level of extracellular alpha-amylase (EC 3.2.1.1) of Bacillus subtilis Marburg was increased about fivefold by introducing the amyR marker from B. natto 1212 through transformation. amyR2 of B. natto 1212 has been assumed to determine a high level of alpha-amylase of the organism. The gene acts specifically on alpha-amylase synthesis but not on the production of other extracellular enzymes. alpha-Amylase of an amyR2-carrying strain was found to be quite similar to that of an isogenic amyR1-carrying strain in the thermostability and electrophoretic behavior of whichever amylase the strain produces. Marburg-type alpha-amylase (amyEm) or B. natto-alpha-amylase (amyEn). Anti-amylase serum titration indicates that a high level of the enzyme activity in the amyR2-carrying strain is caused by the existence of more enzyme rather than the presence of an enzyme having higher efficiency. This is supported further by the fact that amyR controls the synthesis of the amyE gene product in mutant M9, which synthesizes a temperature-sensitive-alpha-amylase, and in mutant M07, which secretes cross-reacting material. The results indicate that amyR regulates the rate of alpha-amylase synthesis.     

Mutation of Bacillus subtilis causing hyperproduction of alpha-amylase and protease, and its synergistic effect.

Mutants that had a genetic lesion increasing the production of alpha-amylase and protease simultaneously were isolated from a transformable strain of Bacillus subtilis Marburg by N-methyl-N'-nitro-N-nitrosoguanidine treatment. These mutants produced two to three times more alpha-amylase and five to 16 times more protease than their parent and were tentatively referred to as AP mutants. As this mutation seems to have occurred at a single gene of the bacterial chromosome and was not located near the alpha-amylase structural gene, the gene was designated as "pap." When pap- and amyR2 (an alpha amylase regulator gene) or pap- and ProH coexisted in the same cell, synergistic effects of the two genetic characters were observed on the alpha-amylase and protease production, respectively. Upon introduction of the pap mutation, the following phenotypic changes were observed in addition to changes in alpha-amylase and protease productivity. (i) Mutants lost the character of competence for the transformation. (ii) When cells were cultured at 30 C for 30 h, mutant cells became filament owing to the formation of chains of cells. (iii) Autolysis of cells was decreased in the mutants. When pap- was transferred to the wild strain by deoxyribonucleic acid-mediated transformation, the transformants showed all these phenotypic alterations simultaneously.     

Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation.

The amyR3, amyS, papS1, tmr, and papM118 mutations each stimulate alpha-amylase production two- to sevenfold above the level in a wild-type strain of Bacillus subtilis. A strain which presumably has all five of these mutations produced 250-fold more alpha-amylase.     

Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation

The amyR3, amyS, papS1, tmr, and papM118 mutations each stimulate alpha-amylase production two- to sevenfold above the level in a wild-type strain of Bacillus subtilis. A strain which presumably has all five of these mutations produced 250-fold more alpha-amylase.     

Cloning and Expression of Thermostable alpha-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

Alpha-amylase genes (amyR2 and amyE+) from an alpha-amylase-hyperproducing Bacillus subtilis strain: molecular cloning and nucleotide sequences.

amyR2, amyE+, and aroI+ alleles from an alpha-amylase-hyperproducing strain, Bacillus subtilis NA64, were cloned in temperate B. subtilis phage p11, and the amyR2 and amyE+ genes were then recloned in plasmid pUB110, which was designated pTUB4. The order of the restriction sites, ClaI-EcoRI-PstI-SalI-SmaI, found in the DNA fragment carrying amyR2 and amyE+ from the phage genome was also found in the 2.3-kilobase insert of pTUB4. Approximately 2,600 base pairs of the DNA nucleotide sequence of the amyR2 and amyE+ gene region in pTUB4 were determined. Starting from an ATG initiator codon, an open reading frame was composed of a total 1,776 base pairs (592 amino acids). Among the 1,776 base pairs, 1,674 (558 amino acids) were found in the cloned DNA fragment, and 102 base pairs (34 amino acids) were in the vector pUB110 DNA. The COOH terminal region of the alpha-amylase of pTUB4 was encoded in pUB110. The electrophoretic mobility in a 7.5% polyacrylamide gel of the alpha-amylase was slightly faster than that of the parental alpha-amylases. The NH2 termination portion of the gene encoded a 41-amino acid-long signal sequence (Ohmura et al., Biochem. Biophys. Res. Commun. 112:687-683, 1983). The DNA sequence of the mature extracellular alpha-amylase, a potential RNA polymerase recognition site and Pribnow box (TTGATAGAGTGATTGTGATAATTTAAAAT), and an AT-rich inverted repeat structure which has free energy of -8.2 kcal/mol (-34.3 kJ/mol) were identified. The AT-rich inverted repeat structure seemed to correspond to the hyperproducing character. The nucleotide sequence around the region was quite different from the promoter region of the B. subtilis 168 alpha-amylase gene which was cloned in the Escherichia coli vector systems.     

Genes affecting the productivity of alpha-amylase in Bacillus subtilis Marburg.

Genetic control of alpha-amylase (alpha-1,4-glucan glucanohydrolase, EC 3.2.1.1.) production by Bacillus subtilis 168 was studied from the standpoint that alpha-amylase production by bacteria is dependent on a long-lived messenger ribonucleic acid and obeys the following equation: E = kappa integral of X-DT where x = cell mass at time t, E = alpha amylase produced, t = culture time, and kappa = productivity constant. So a productivity constand (kappa) is obtained from the slope of the straight line plot of alpha-amylase formed versus the total mass of cells accumulated over that time during the culture process. The following results were obtained. (i) Two sequential mutants, derived from the 168(kappa = 20) strain and having improved alpha-amylase productivity (168 leads to 196), were analyzed for their serine and metal protease production. Strain 128 (kappa = 40) produced half the amount of both proteases, but strain 196 (kappa = 60 similar to 80) produced 20 times that in the original strain. (ii) Amy+ transformants, using the 196 strain as the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71, J47, groups. Seventy-one of 74 Amy+ transformants had a kappa value of 21.0 plus or minus 2.1 and the other three had higher productivity (kappa = 37 similar to 46). These transformants (J71,J47, and J10) produced levels of serine and metal proteases 20 times higher than the other transformants. (iii) Strains 196, J71, J47, and J10 were found to be nonmotile and resistant to phage PBS1, whereas other strains, including strains 168, 128, 3 revertants of strain J71 and 2 revertants of strain 196, were all motile and sensitive to the phage. (iv) Strains 196 and J71 were nonflagellated under electron microscopic observation but strain 168, 128 and a revertant of J71 were flagellated. From the above experimental results, the existence of a quality controlling gene (amyB) was deduced, which is loosely linked to the structural gene and controls productivities of alpha-amylase and proteases, and flagellation. The probable existence of another regulatory gene, amyC, is also discussed.     

Molecular cloning of cis-acting regulatory alleles of the Bacillus subtilis amyR region by using gene conversion transformation.

Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Regulation of Neutral Protease Productivity in Bacillus subtilis: Transformation of High Protease Productivity

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

Isolation of Mutants Defective in α-Amylase from Bacillus subtilis: Genetic Analyses

The rate of alpha-amylase (EC 3.2.1.1) synthesis in Bacillus subtilis is regulated by a gene, amyR, located near a structural gene, amyE, for the enzyme. To construct a fine map of the amyR-amyE region, we isolated 28 mutants defective in alpha-amylase activity. Eleven mutants out of 28 showed no alpha-amylase activity, whereas the other 17 showed less alpha-amylase activity than the parent. Out of 17 partially positive alpha-amylase mutants, 10 produced temperature-sensitive enzymes, and 4 produced immunologically altered enzymes, two of which are concurrently temperature-sensitive, and 5 produced smaller amounts of alpha-amylases which are indistinguishable from normal enzyme in their temperature sensitivity and immunological properties. Two out of 11 alpha-amylase-negative mutants produced material that cross-reacted with anti-amylase serum, and 3 mutants carried suppressible mutations by the suppressor described by Okubo. Mapping data indicate that all 28 mutation sites are located in the amyE region, and none of the groups of the mutants mentioned above contains lesions that are clustered in a single region of amyE. The amyR gene seems most likely to adjoin the terminal region of amyE.     

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.     

Designed gene amplification on the Bacillus subtilis chromosome

We previously reported the cloning of a 1.6 kb HindIII fragment (containing the junction of the repeating unit) from chromosomal DNA of Bacillus subtilis strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the B. subtilis chromosome, a gene amplification of the 22 kb repeating unit containing the alpha-amylase structural gene (amyE), the tunicamycin-resistance gene (tmrB) and the shikimate kinase structural gene (aroI). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a B. subtilis wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tmr) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tmr transformants showed high productivity of alpha-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10-20. This system may provide an effective means of amplifying long (greater than 20 kb) DNA regions on the chromosome.     

Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated

A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis.

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Comparison of the α-Amylase of Bacillus subtilis and Bacillus amyloliquefaciens

The alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) of Bacillus subtilis strain W23 is less negatively-charged than the alpha-amylase of B. amyloliquefaciens strain F, as determined by electrophoretic mobility in polyacrylamide gel at pH 8.6. The alpha-amylase of strain W23 is immunologically unrelated to the alpha-amylase of strain F, as judged by lack of cross-reaction in Ouchterlony immunodiffusion studies. The pH range of maximal activity for the enzyme of strain W23 was 5.7 to 6.7, with a maximum at 6.3. The pH range of activity for the alpha-amylase of strain F was 5.5 to 6.5, with a maximum at 5.9. No significant difference was found in the effect of temperature on the activity of the alpha-amylase of strain W23 and strain F. alpha-Amylase production by strain W23 occurs throughout the 7-hr growth period, whereas enzyme production by strain F does not begin until the culture enters the stationary phase of growth. The total amounts of enzyme produced by strains W23 and F after 7 hr of growth were 0.3 and 25.5 units/ml, respectively.