High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

By marker exchange mutagenesis, Bacillus megaterium strain UN-1 (Bm-UN1) was used to prepare a mutant strain B. megaterium UN-cat (Bm-UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm-UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm-UNcat and Bacillus subtilis. Bm-UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13.7, 19.5 and 20.4 U ml(-1) at 24, 36 and 48 h of culture, respectively. This was two- to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20-fold higher than PAC produced by the parent strain, Bm-UN1.     

Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity

Chitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10 mU of this chitinase, the LD(50) of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10 mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5alpha and sequenced to reveal a single open reading frame of 1,815 bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15 mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of two beta-xylosidase genes of Bacillus pumilus and comparison of their gene products

The chromosomal DNA fragments of Bacillus pumilus IPO, a potent xylan-hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transform Escherichia coli C600 cells. Two hybrid plasmids, pOXD28 and pOXN29, were found to enable the transformants to produce beta-xylosidase. The former was found to contain a 2.6-MDa Bg/II fragment and the latter, a 7.7-MDa PstI fragment, both coding beta-xylosidase, but xylanase is coded only on the latter hybrid plasmid. The DNAs inserted in both plasmids originated from the B. pumilus chromosome, but from different regions, as shown by Southern hybridization and the analysis of restriction fragments. beta-Xylosidases I and II, coded on pOXN29 and pOXD28 respectively, were purified to homogeneous preparations and compared. Both were dimer enzymes consisting of 65000-70000-Da subunits. Specific activity and the Km value of beta-xylosidase I to p-nitrophenyl beta-D-xyloside as substrate were respectively 100 and 1/40 times those of beta-xylosidase II. The mobilities of beta-xylosidases I and II on polyacrylamide gel electrophoresis were also different. beta-Xylosidase I, the gene of which is located near the xylanase gene on pOXN29, can convert xylooligosaccharides to xylose, but beta-xylosidase II had little activity on xylobiose. These results suggest that beta-xylosidase I is the main enzyme for xylan hydrolysis in B. pumilus.     

Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli

Summary The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and -xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and -xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and -xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the -xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not -xylosidase, was secreted into the medium in a B. pumilus culture.     

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC₅₀s were 5.6 × 10⁴ and 2.4 × 10⁵ spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.