Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Cloning of the glycerol kinase gene of Bacillus subtilis

A 3.5 kb fragment of Bacillus subtilis DNA which contains wild type alleles of mutations in glpK (glycerol kinase) and glpD (glycerol-3-phosphate [G3P] dehydrogenase) was cloned in plasmid pHV32 in Escherichia coli. The cloned fragment expresses glycerol kinase in B. subtilis mutants carrying the mutations glpK11 and recE4 after induction with glycerol or G3P whereas it does not express G3P dehydrogenase. The cloned fragment thus contains the complete glpK but probably only part of glpD.     

枯草杆菌表达载体pUB23的构建及地衣杆菌α-淀粉酶基因的表达

将克隆的解淀粉芽胞杆菌强启动子经DNA序列分析后连接到能在枯草杆菌中复制的质粒pUB18上,构建枯草杆菌表达载体pUB23。为了测试构建的表达载体能否表达外源基因,将地衣杆菌抉失了启动子的α-淀粉酶基因接到pUB23上启动子的下游,组建重组质粒,转化枯草杆菌QB1130(amy~-),获得能分泌α-淀粉酶的转化株,证明缺失了启动子的结构基因在pUB23上克隆启动子的启动下获得表达。酶活力测定结果表明,表达水平是用原启动子时的2.5倍.     

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.     

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2.

By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a lambda gt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA. Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene. The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da. When expressed in Escherichia coli DH5 alpha, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE. The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCl2-dependent (20 mM optimum) and LiCl-sensitive. The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgCl2-dependent. Initial mapping experiments located the autolysin gene near aroD on the B. subtilis 168 chromosome.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

环状芽孢杆菌α—羟基—γ—氨丁酰酰化酶基因的克隆和表达

本文报道了以质粒pUB110为载体,以枯草芽孢杆菌(Bacillus subtilis 168)作为受体菌,对丁酰苷菌素产生菌(Bacillus circulans NRRL-B3312)总DNA进行了鸟枪克隆,在所获得的转化子中,No.733转化子经薄层层析,生物显迹和质谱分析表明,它具有将卡那霉素A生物转化成为丁胺卡那霉素的能力,说明该转化子所含重组质粒pUBC733的插入片段中含有a-羟基-r-氨丁酰(HABA)酰化酶基因,HABA酰化酶基因已经在枯草芽孢杆菌中获得了克隆和表达。该重组质粒分子量为7.3kb,插入片段为2.8kb,经Southern分子杂交确证此片段确来源于环状芽孢杆菌,已构建了该质粒限制性内切酶图谱。     

Molecular cloning and purification of klebicin B

A novel klebicin, klebicin B, produced by an isolate of Klebsiella pneumoniae has been identified. It is encoded by a 5.5 kb plasmid, pKlebB-K17/80, which is mobilized into K. pneumoniae UNF5023 by a large plasmid found in the same strain. The 5.5 kb plasmid has been cloned into the high-copy-number vector pUC19 and the restriction map of the resulting recombinant plasmid pRJ180 has been determined. Using sub-cloning and transposon mutagenesis, the klebicin B structural gene, the klebicin B immunity gene and the mitomycin C (MC) sensitivity gene (lys) present on pRJ180 have been localized. Transposon inserts which inactivated klebicin production also abolished lysis protein production encoded by pRJ180, but did not affect klebicin B immunity. Using SDS-PAGE an MC-induced polypeptide of 85 kDa was observed in cultures of K. pneumoniae UNF5023(pRJ180). This polypeptide was absent in cultures carrying plasmid pRJ180 with a Tn1000 insert which inactivated klebicin production. Analysis of the polypeptides present in the medium of Escherichia coli JM83 hsdR(pRJ180) or K. pneumoniae UNF5023(pRJ180) indicated that the 85 kDa polypeptide is specifically secreted from the producing cell. Klebicin B has been purified, using gel filtration, from a cell-free extract of K. pneumoniae UNF5023(pRJ180) which had been induced with MC. After boiling in sample buffer the purified klebicin B gave rise to two peptides on SDS-PAGE, one of 85 kDa and the other of 11 kDa. Klebicin B-resistant mutants of K. pneumoniae UNF5023 were sensitive to klebicin A, colicin B and colicin D.     

Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth

A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B. subtilis mutant temperature-sensitive in spore outgrowth. The cloned region is a single EcoRI fragment 14 kb in length. In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci.     

Multiple endo-beta-1,4-glucanase-encoding genes from Bacillus lautus PL236 and characterization of the celB gene

A Bacillus lautus strain was isolated from compost by its ability to degrade microcrystalline Avicel cellulose and acid-swollen cellulose. Three DNA fragments cloned in Escherichia coli encoded at least four endo-beta-1,4-glucanases (EG), of which at least two were contained on one DNA fragment. Another fragment, of 2.5 kb and carrying celB, was cloned in the shuttle-vector plasmid, pJKK3-1, and expressed in E. coli and Bacillus subtilis. The fragment was sequenced and shown to encode a 62-kDa protein, which was found as a 56-kDa mature and active EG in extracts of E. coli and in the supernatant of B. subtilis. The deduced amino acid (aa) sequence has a homology of 37% identical aa on a stretch of 295 aa to EG-E of Clostridium thermocellum. A low level of homology is detected with the Bacillus-type EG.     

Molecular analysis of the tagF gene, encoding CDP-Glycerol:Poly(glycerophosphate) glycerophosphotransferase of Staphylococcus epidermidis ATCC 14990

Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168. The S. epidermidis tagF gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into B. subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned S. epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike B. subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S. epidermidis is not linked to any other tag genes. We attempted to disrupt the chromosomal tagF gene in S. epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5' end of tagF on a similarly sized DNA fragment was able to integrate. This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.     

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.     

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.