Functional properties of the pOV13 plasmid as a vector for DNA cloning in a broad spectrum of gram negative bacteria

The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

Homologous sequences other than insertion elements can serve as recombination sites in plasmid drug resistance gene amplification.

A plasmid (pRR983) was constructed which has a gene coding for neomycin and kanamycin resistance flanked by direct repeats of regions of homology which contain no known insertion sequences. pRR983 does not have any homologous IS1 sequences. Growth of Proteus mirabilis harboring pRR983 in medium containing high concentration of neomycin resulted in cells which were highly resistant to both neomycin and kanamycin. Plasmid DNA was analyzed by using restriction endonucleases. In most cases the neomycin resistance gene had been tandemly duplicated by using the homologous DNA sequences flanking the resistance gene as recombination sites. This is analogous to tandem duplication of drug resistance genes on NR1 using the two direct repeats of IS1 as recombination sites. The amplified plasmid DNA returned to its original structure by the deletion of amplified neomycin resistance determinants when the host cells were cultured without selection for high resistance to neomycin.     

Construction and use ofAgrobacterium tumefaciens binary vectors withA. tumefaciens C58 T-DNA genes

Five plant morphoregulatory genes were isolated from the Agrobacterium tumefaciens Ti plasmid and binary plasmid vectors for plant transformation with these genes were constructed. All vectors have a similar structure with T-DNA borders, RK2 origin of replication and chimeric kanamycin resistance gene for the selection of transformed plant tissues. Over twenty vectors with single and combined morphoregulatory genes were constructed and their effects after tobacco tissue transformation studied.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Marker Removal in Staphylococci via Cre Recombinase and Different lox Sites

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and S. aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

Integration of R plasmid Rts1 to the gal region of the Escherichia coli chromosome.

An R plasmid Rts1 was integrated into the gal region of the chromosome of Escherichia coli XA-7012 (galE) strain by the directed transposition technique. The integration of the Rts1 genome was confirmed mainly by conjugation studies and also by transduction experiments using phage P1. As a result, it was found that the integrated genome contained genes responsible for kanamycin resistance, conjugal transferability, and for autonomous replication. As reported previously, Rts1 is temperature sensitive in replication and inhibits the growth of the host at nonpermissive temperature. However, although a plasmid derived from the integrated Rts1 genome still demonstrates temperature sensitivity upon transfer and high level of kanamycin resistance, this plasmid no longer displays temperature sensitivity in replication and the inhibitory effect on the host. These results indicate that the temperature sensitivity of replication of Rts1 and its inhibitory effect on the host cell are due to the presence of a gene or gene cluster on the Rts1 genome and that the gene(s) is clearly discriminated from the one responsible for the temperature sensitivity of transfer.     

弗氏志贺菌 M90T 株毒力相关膜蛋白复合物组成基因缺失突变体的构建

目的：构建弗氏志贺菌M90T株毒力相关膜蛋白复合物组成蛋白基因的缺失突变株。方法：分别扩增目的基因的上、下游同源臂和卡那霉素抗性基因片段,利用重叠延伸PCR技术将这3个片段融合为打靶片段,电击转化M90T／pKD46感受态细胞,在bRed重组系统的作用下,通过同源重组将目的基因置换为卡那霉素抗性基因,之后在辅助质粒pCP20的作用下切除FRT位点之间的卡那霉素抗性基因,得到基因缺失突变体。结果与结论：分别构建了M90T株毒力相关膜蛋白复合物4个组成蛋白Apy、DnaK、CIpB、YdgA的基因缺失突变体,为进一步研究各基因的功能提供了突变体。     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Chloramphenicol acetyltransferase expression in marine Rhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

A vector, pUK318, was constructed to allow the expression of foreign genes in the marine photosynthetic bacterium Rhodobacter sp. NKPB 0021. This strain has been cured of its two endogenous plasmids. pUK318 consists of a 2.3-kb PstI-BamHI restriction fragment, containing a marine Rhodobacter plasmid replication region, cloned into pUC18. This fragment was derived from plasmid pRD31, a 3.1-kb endogenous plasmid purified from the marine strain Rhodobacter sp. NKPB 043402. A kanamycin resistance gene from Tn903 was cloned into the PstI restriction site to provide antibiotic selection. pUK318 was transferred to Rhodobacter sp. NKPB 0021 by transformation, and efficiencies of 7.2 x 10(-5) were obtained. Furthermore, pUK318 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics. pUK318 was used to express the Escherichia coli chloramphenicol acetyl transferase (CAT) gene in Rb. NKPB 0021. Transformants expressed a maximum CAT activity of 1.12 mmol/min/g dry cells. In addition, the DNA region essential for pUK318 replication in Rb. NKPB 0021 was localized to a 1.36-kb HincII-PstI fragment. This is the first report of a plasmid vector containing a marine Rhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection.     

增加复制起始区对基因表达的影响

我们在质粒ｐｕＢ１１０的基础上组建了ｐＤＲ质粒,它们具有双复制起始区而只有一个抗卡那霉素基因。携带了这些质粒的宿主细胞对卡那霉素的抗性明显高于亲本株（Ｂ,ｓｕｂｔｉｌｉｓ １５０（ｐｕＢ１１０））。经限制性酶切图谱分析新获得的转化株具有二个复制起始区及一个 Ｋｍ＆４基因。说明增加复制起始区是提高重组子表达能力的途径之一。     

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).     

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.     

A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)

A set of plasmid vectors conferring chloramphenicol resistance (Cm(R)), 3064bp in size, or kanamycin resistance (Km(R)), 2972bp in size, were developed, having multiple cloning sites in lacZ' genes for alpha-complementation. pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 are temperature-sensitive (ts) in DNA replication (ts-Rep); pTH18cs5, pTH19cs5, pTH18ks5 and pTH19ks5 are ts in plasmid segregation (ts-Seg); and pTH18cr, pTH19cr, pTH18kr and pTH19kr are temperature resistant (tr) in both. They are based on the pSC101 replicon consisting merely of the replication origin and repA gene, compatible with ColE1/pMB1/p15-derived plasmids, and thus do not require polA function of host cells. The copy numbers of the ts-Rep, tr and ts-Seg plasmids were 14, 5 and 1 per chromosome at 30 degrees C, respectively. These plasmids are fairly stable when inherited at 30 degrees C, but not above 37 degrees C or 41.5 degrees C, depending on the repA mutations and host strains. They are isogenic apart from the ts mutations in the repA gene, and thus provide with useful tools for having appropriate controls in various experiments including bacterial gene-targeting, transposon mutagenesis, toxic gene expression, differential substitution on host functions, gene dosage analysis and so on.