Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

Molecular cloning of the structural gene for Acinetobacter citrate synthase

The structural gene for citrate synthase of Acinetobacter anitratum has been cloned in Escherichia coli in a form which expresses the enzyme. A library of EcoRI fragments of Acinetobacter genomic DNA was prepared in the vector lambda gt10, and clones were screened by hybridization with an E. coli citrate synthase clone under conditions of reduced stringency. A 6.5 kbp clone was obtained which was subcloned into pBR322, and shown to direct the formation of Acinetobacter citrate synthase in E. coli hosts. The promoter was located within a BglII fragment, and from this information the orientation of the gene was deduced.     

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.     

Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens

Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B.

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli

A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).