Cloning and overexpression of <Emphasis Type="Italic">aprE3-17</Emphasis> encoding the major fibrinolytic protease of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> CH 3-17

Bacillus licheniformis (B. licheniformis) CH3-17, an isolate from cheonggukjang, a traditional Korean fermented soyfood, secretes several fibrinolytic enzymes into the culture medium, showing strong fibrinolytic activity. A gene homologous to aprE of Bacillus subtilis (B. subtilis), aprE3-17, was cloned by PCR. DNA sequencing showed that aprE3-17 encodes a prepro-type serine protease consisting of 382 amino acids. The mature enzyme was 27 kDa in size. The aprE3-17 gene was overexpressed in B. subtilis WB600 using pHY300PLK, an Escherichia coli (E. coli)-Bacillus shuttle vector, and the 27 kDa enzyme was purified from the culture supernatant. The optimum pH for activity was 6.0. Purified enzyme quickly degraded the Aα and Bβ chains of fibrinogen but could not degrade the γ-chain.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Application of the Upstream Region of a Bacillus Endoglucanase Gene to High-level Expression of Foreign Genes in Bacillus subtilis

A 0.4-kb ScaI-HpaI fragment, 199bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5′ region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

Cloning and expression of a bpr gene encoding Bacillopeptidase F from Bacillus amyloliquefaciens CH86-1

A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.     

Gene Cloning,Expression, and Properties of a Fibrinolytic Enzyme Secreted by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> BS15 Isolated from Gul (Oyster) Jeotgal

A Bacillus strain, BS15, showing strong fibrinolytic activity, antibacterial activity, and salt tolerance was isolated from gul (oyster) jeotgal, a Korean fermented sea food. BS15 was identified as B. pumilus. B. pumilus BS15 was able to grow in LB broth with 18% (w/v) NaCl. When culture supernatant was analyzed by SDS-PAGE, 22, 27, 35, and 60 kDa proteins were observed. The 27 kDa protein was determined to be major fibrinolytic enzyme by fibrin zymography. The gene (aprEBS15) was cloned in pHY300PLK, a Bacillus-E. coli shuttle vector. A B. subtilis transformant (TF) harboring pHYBS15 showed higher fibrinolytic activity than B. pumilus BS15, and produced the same 27 kDa protein. aprEBS15 was overexpressed in E. coli BL21 (DE3), and recombinant enzyme (AprEBS15) was purified. The optimum pH and temperature of AprEBS15 were pH 8.0 and 40°C, respectively. Km and Vmax values were 0.26 mM and 21.88 µmol/L/min, respectively. B. pumilus BS15 can be used as a starter for jeotgals and other fermented foods with high salinities.     

Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+-ATPase by cloned genes from the same and different species

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha-subunit gene of the F₁F₀ type of H⁺-ATPase was deleted. MS117 had low H⁺-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha-subunit gene of E. hirae H⁺-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F₁F₀-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H⁺-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F₁F₀ type of H⁺-ATPase.     

Industrial applications of a cloned neutral protease gene in Bacillus subtilis

Summary Genes for the -amylase and neutral protease were cloned from an industrial Bacillus isolate, Bsl, onto two separate plasmids and introduced into a B. subtilis strain. Both plasmids were stably maintained in this strain. Analysis of the extracellular proteins showed that the plasmidcarrying strain produced predominantly the Bsl -amylase and neutral protease with few contaminating B. subtilis exoenzymes. The presence of high levels of protease enabled the strain to produce considerably more -amylase when grown on a complex industrial medium rich in protein.     

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.     

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria

The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 g ml^-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromsomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromsomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.     

Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. aizawai toward Lymantria dispar larvae

Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl -D-N,N- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae (Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.     

Cloning,Sequencing, and Expression of a β-Amylase Gene from Bacillus cereus var. mycoides and Characterization of Its Products

The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known β-amylases. The N-terminal sequence of the cloned β-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.     

Enhancement of the antifungal activity of Bacillus subtilis F29-3 by the chitinase encoded by Bacillus circulans chiA gene

Bacillus subtilis F29-3 is an antagonistic bacterium against a wide range of fungal species. In order to determine the effect of chitinase on the antifungal activity of B. subtilis F29-3, a 2.4-kb DNA fragment containing the chiA gene of Bacillus circulans WL-12 was ligated into a shuttle vector pHY300PLK and transformed into B. subtilis F29-3. A bioassay conducted on the culture supernatant showed that, in comparison to the B. subtilis control strain, B. subtilis F29-3 expressing the chiA gene exhibited a greater inhibition of spore germination of Botrytis elliptica, indicating that chitinase could enhance the antifungal function conferred by B. subtilis F29-3.     

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.     

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     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis

Summary The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the -amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-polyacrylamide gel electrophoresis characterizations. The NH₂-terminal sequence analysis of the secreted PME revealed two different NH₂-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and -amylase signal peptide. Offprint requests to: R. Heikinheimo     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.