Cloning and expression of the Escherichia coli recA gene in Bacillus subtilis

By means of homopolymer dG-dC tailing, using PstI linearized pBR327 as vector, we constructed small plasmids containing the entire Escherichia coli recA gene. The 1.8-kb inserts were recloned in the Bacillus subtilis expression vector pPL608 in a B. subtilis recE4 strain. Analysis of plasmid-coded proteins showed expression of the E. coli recA gene both in minicells and whole cells of B. subtilis. Expression was under control of the bacteriophage SP02 promoter, which is part of pPL608. A recA-expressing plasmid completely abolished the transformation deficiency of the recE4 mutant as well as its sensitivity to mitomycin C (MC). The expressed recA gene also restored recombination in other B. subtilis strains lacking the recE gene product. These results indicate a high similarity between the functions of the E. coli RecA and B. subtilis RecE proteins.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.     

Cloning and expression of the Bacillus subtilis methyltransferase gene in Escherichia coli ada- cells

DNA fragments of Bacillus subtilis were inserted into a plasmid vector that can multiply in Escherichia coli cells, and foreign genes were expressed under the control of the lac promoter. By selecting hybrid plasmids that confer an increased resistance to alkylating agents on E. coli ada- mutant cells, the B. subtilis gene dat, which encodes O6-methylguanine-DNA methyltransferase, was cloned. The Dat protein, with a molecular weight of about 20,000, could transfer the methyl group from methylated DNA to its own protein molecule. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is 18,779. The presumptive amino acid sequence of Dat protein is homologous to the sequences of the E. coli Ogt protein and the C-terminal half of the Ada protein, both of which carry O6-methylguanine-DNA methyltransferase activity. The pentaamino acid sequence Pro-Cys-His-Arg-Val, the cysteine residue of which is the methyl acceptor site in Ada protein, was conserved in the 3 methyltransferase proteins. The structural similarity of these methyltransferases suggests possible evolution from a single ancestral gene.     

Cloning the Escherichia coli K-12 argD gene specifying acetylornithine delta-transaminase

The argD gene of Escherichia coli was shown to be present in plasmids pLC2-28 and pLC3-11 of the collection of Clarke and Carbon [Cell 9 (1976) 91-99]. The gene was cloned into pBR322 as a 6.3-kb BamHI fragment. Enzyme determination showed that the cloned DNA contains the structural gene for acetylornithine delta-transaminase. The argD DNA was used as a probe in hybridization experiments which indicated that the argM gene resides in a duplicated portion of E. coli DNA that is highly similar to the argD region.     

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

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.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Transformation in Bacillus subtilis: involvement of the 17-kilodalton DNA-entry nuclease and the competence-specific 18-kilodalton protein.

A protein complex, consisting of a 17-kilodalton (kDa) nuclease and an 18-kDa protein, is believed to be involved in the binding and entry of donor DNA during transformation of Bacillus subtilis (H. Smith, K. Wiersman, S. Bron, and G. Venema, J. Bacteriol. 156:101-108, 1983). In this paper, the nucleotide sequences of the genes encoding both the nuclease and the 18-kDa protein are presented. The genes are encoded by a 904-base-pair PstI-HindIII fragment. The open reading frames encoding both proteins are partly overlapping. A B. subtilis mutant was constructed by insertion of a Cmr marker into the gene encoding the nuclease. This mutant lacked the competence-specific nuclease activity and the 18-kDa protein but retained 5% residual transformation. The total DNA association of the mutant was higher than that of the wild-type cells, and DNA entry was reduced to 30% of the wild-type level. These results suggest that an alternative pathway exists for the internalization of transforming DNA. A mutant, exclusively deficient for the 18-kDa protein, previously suggested to be involved in the binding of transforming DNA, was constructed by insertion of a kanamycin resistance gene into the coding sequence of the gene. Since the mutant showed wild-type DNA-binding activity, the 18-kDa protein is probably not involved in the binding of donor DNA to competent cells. The transforming activity of the mutant was reduced to 25% of the wild-type level, indicating that the 18-kDa protein has a function in the transformation process. In vitro experiments showed that the 18-kDa protein is capable of inhibiting the activity of the competence-specific nuclease. Its possible role in transformation is discussed.     

Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis.

A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E. coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E. coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.     

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.     

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1% xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an Mr of 22 000.     

Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli

The gene coding for the subunits of aspartokinase II from Bacillus subtilis has been identified in a B. subtilis DNA library and cloned in a bacterial plasmid (Bondaryk, R. P., and Paulus, H. (1984) J. Biol. Chem. 259, 585-591). The introduction of a plasmid carrying the aspartokinase II gene into an auxotrophic Escherichia coli strain lacking all three aspartokinases restored its ability to grow in the absence of L-lysine, L-threonine, and L-methionine. The B. subtilis aspartokinase gene could thus be functionally expressed in E. coli and substitute for the E. coli aspartokinases. Measurement of aspartokinase levels in extracts of aspartokinaseless E. coli transformed with the B. subtilis aspartokinase II gene revealed an enzyme level comparable to that in a genetically derepressed B. subtilis strain. In spite of the high level of aspartokinase, the growth of the transformed E. coli strain was severely inhibited by the addition of L-lysine but could be restored by also adding L-homoserine. This apparently paradoxical sensitivity to lysine was due to the allosteric inhibition of B. subtilis aspartokinase II by that amino acid, a property which was also observed in extracts of the transformed E. coli strain. The synthesis and degradation of the aspartokinase II subunits were measured by labeling experiments in E. coli transformed with the B. subtilis aspartokinase II gene. In contrast to exponentially growing cells of B. subtilis which contained equimolar amounts of the aspartokinase alpha and beta subunits, the transformed E. coli strain contained a 3-fold molar excess of beta subunit. Pulse-chase experiments showed that the disproportionate level of beta subunit was not due to more rapid turnover of alpha subunit, both subunits being quite stable, but presumably to a more rapid rate of synthesis. After the addition of rifampicin, the synthesis of alpha subunit declined much more rapidly than that of beta subunit, indicating that the two subunits were translated independently from mRNA species that differ in functional stability. In conjunction with the results described in the preceding paper which demonstrated that the aspartokinase subunits are encoded by a single DNA sequence, these observations imply that the alpha and beta subunits of B. subtilis aspartokinase II are the products of in-phase overlapping genes.     

Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis.

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.