Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.     

Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum

Novel cloning vectors for glutamic acid-producing bacteria have been constructed. Two cryptic plasmids, pAM330 from Brevibacterium lactofermentum and pHM1519 from Corynebacterium glutamicum, were used as precursors, and recombined with pBR325 or pUB110. Resultant composite plasmids were able to propagate and to express the CmR or KmR phenotype in B. lactofermentum and C. glutamicum. A smaller, high-copy-number plasmid, pAJ43, was also isolated following deletion of a part of the pAM330-pBR325 composite plasmid. Furthermore, a cosmid vector, which can be packaged and transduced through phage infection, has been developed using a cohesive-end fragment of the f1A phage and plasmid pAJ43. These plasmids are suitable for use as cloning vectors in the glutamic acid-producing bacteria.     

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.     

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.     

Suicide vector for transposon mutagenesis in Pseudomonas solanacearum.

A suicide vector was constructed by cloning the transfer genes of the wide-host-range (IncW group) plasmid R388 into the BamHI site of pBR325. This plasmid can deliver Tn5 into Pseudomonas solanacearum at frequencies ranging from 10(-6) to 10(-9) per recipient.     

High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection

Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

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.     

Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector.

We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. Both antibiotic resistance genes are efficiently expressed in C. michiganense subsp. michiganense. Although polyethylene glycol-mediated transfection of spheroplasts with the DNA of the C. michiganense subsp. michiganense-specific bacteriophage CMP1 yielded about 3 x 10(3) transfectants per microgram of DNA, in transformations with plasmid DNA only a very few transformants were obtained. However, the transformation efficiency could be improved by electroporation of intact cells, giving about 2 x 10(3) transformants per microgram of plasmid DNA. Since a transformation procedure and a cloning vector are now available, pathogenicity in C. michiganense subsp. michiganense can now be analyzed genetically.     

Expression and stability of a recombinant plasmid in Zymomonas mobilis and Escherichia coli

A recombinant plasmid was constructed by ligating EcoRI digests of the plasmid cloning vector pBR325 and pZMO2, one of the natural plasmids of Zymomonas mobilis ATCC 10988. This vector, named pDS212 (total size 7.9 kb), which was able to transform Escherichia coli efficiently, was also transferred to Z. mobilis hosts by mobilization during conjugation using the helper plasmid pRK2013. pDS212 was inherited stably in both E. coli and Z. mobilis hosts and could be recovered intact from them. Markers of pBR325 and pRK2013 were also transferred in Z. mobilis but at very low frequencies. Neither pBR325 nor pRK2013 could be recovered intact from the Z. mobilis hosts. It is proposed that expression and stability of pDS212 in Z. mobilis is due to the origin of replication of pZMO2 that it carries, and that it may be used for developing a gene transfer system in Z. mobilis.     

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

A modified pBR322 vector with improved properties for the cloning, recovery, and sequencing of blunt-ended DNA fragments

The construction of a plasmid vector which facilitates the cloning and recovery of blunt-ended DNA fragments is described. This plasmid, called pHP34, differs from pBR322 by a 10-bp insertion which introduces a unique SmaI site immediately flanked by two EcoRI sites. Blunt-ended DNA fragments cloned in the SmaI site can be recovered by digestion with EcoRI. Small cloned fragments can be chemically sequenced using a strategy which does not require their purification. The use of a plasmid related to pHP34 for in vitro mutagenesis by the insertion of a DNA linker fragment conferring an antibiotic resistance is also discussed.     

Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants.

A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.     

Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

We used a modified version of the method of Hanahan (D. Hanahan, J. Mol. Biol. 166:557-580, 1983) to transform Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica with the plasmids pBR322, pBR325, and pAT153. The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA. The nature of these transformants was confirmed by plasmid analysis. ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species.     

Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives.

A family of cloning vectors derived from plasmid pACYC184 and, therefore, compatible with pBR322 and its derivatives (especially the pUC family of vectors), is described. They all contain a multiple cloning site (MCS) and the lacZ alpha reporter gene for easy cloning. They have been grouped in three sets: (i) six of the vectors contain a chloramphenicol-resistance (CmR)-encoding gene and each a different MCS with 16 unique restriction sites overall; (ii) another six vectors contain a kanamycin-resistance (KmR)-encoding gene and the same six MCS; and (iii) two CmR vectors that contain the SP6 and T7 promoters flanking the MCS and lacZ alpha reporter gene of pUC18/19.     

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.     

Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat

Summary The maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight.Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.     

Construction of a vector for cloning promoters in Bacillus subtilis

A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.