Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.     

Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi

Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non-infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.     

棒杆菌质粒pXZ10145复制功能区定位

将棒杆菌质粒pXZ10145或pNAT65的不同酶切片段装入大肠杆菌质粒pACYCl77中构建了pTSK系列重组质粒。转化棒状类细菌的实验结果确定了质粒pXZ10145上复制必需区的位置。质粒pXZ10145复制最小必需区定位在NaeI-NruI的1.2kb片段上,在这个片段上只有一个约940碱基的阅读框架。它编码一个质粒复制因子,以对位作用方式协助那些不能自我复制但复制起始区仍保持完整的pTSK质粒在棒状类细菌中复制。质粒pXZ10145复制起始区在一个NaeI-SalI的0.3kb片段上,位于已确定的复制因子编码框架中。     

Characterization of replication and conjugation of Streptomyces circular plasmids pFP1 and pFP11 and their ability to propagate in linear mode with artificially attached telomeres

Many Streptomyces species harbor circular plasmids (8 to 31 kb) as well as linear plasmids (12 to 1,700 kb). We report the characterization of two newly detected circular plasmids, pFP11 (35,139 bp) and pFP1 (39,360 bp). As on linear plasmids, their replication loci comprise repA genes and adjacent iterons, to which RepA proteins bind specifically in vitro. Plasmids containing the minimal iterons plus the repA locus of pFP11 were inherited extremely unstably; par and additional loci were required for stable inheritance. Surprisingly, plasmids containing replication loci from pFP11 or Streptomyces circular plasmid SCP2 but not from pFP1, SLP1, or pIJ101 propagated in a stable linear mode when the telomeres of a linear plasmid were attached. These results indicate bidirectional replication for pFP11 and SCP2. Both pFP11 and pFP1 contain, for plasmid transfer, a major functional traB gene (encoding a DNA translocase typical for Streptomyces plasmids) as well as, surprisingly, a putative traA gene (encoding a DNA nickase, characteristic of single-stranded DNA transfer of gram-negative plasmids), but this did not appear to be functional, at least in isolation.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

The plasmid-maintenance functions of the P7 prophage

The region responsible for the maintenance of the prophage of bacteriophage P7 as a stable, unit-copy plasmid was isolated in a lambda att vector which lysogenizes Escherichia coli as a stable unit-copy plasmid under the control of the P7 replication origin. The P7 plasmid-maintenance region was shown to consist of adjacent replication and partition regions capable of functioning independently. The isolated replication region could support plasmid maintenance but the resulting plasmids were highly unstable unless the partition region was also included. Stable composite plasmids were isolated containing the putative P7 partition region and the origin of replication of the unrelated plasmid F, indicating that P7 encoded an active partition mechanism. The replication regions of P7 and P1 were shown to be highly homologous but the partition regions of the two plasmids appear to be unrelated in sequence. The incompatibility determinants associated with the two replication regions showed the same specificity, whereas the partition-region incompatibility determinants were different, showing no cross-specificity.     

A set of plasmids constitutively producing different RNA levels in Escherichia coli.

New plasmids were developed for the in vivo expression of RNA in Escherichia coli. These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA. The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions. The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors. Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter.     

Analysis of a recessive plasmid copy number mutant: evidence for negative control of Col E1 replication.

The Col E1-derivative copy number mutant plasmid pOP1Δ6 has been used to investigate the control of plasmid replication. pOP1Δ6 normally exists at about 200 copies per chromosome, while the wild-type plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1Δ6 and pBGP120, the copy number of pOP1Δ6 is lowered to 4–6 copies per chromosome. Thus the mutation in pOP1Δ6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOP1Δ6. Co-residence of the unrelated plasmid pSC101 with pOP1Δ6 has no detectable effect on pOP1Δ6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOP1Δ6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQP1Δ6 but contains a replication origin region derived from pBGP120. Since this plasmid, pNOP1, exists stably (like pBGP120) at 10–15 copies per chromosome, the high copy number of pOP1Δ6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOP1Δ6 responsible for DNA overproduction, we have cloned fragments of pBGP120 into pOP1Δ6 and selected for plasmids with wild-type copy number. We find that a 2.0 kb region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOP1Δ6. The 2.0 kb fragment is capable of independent self-replication or can integrate into pOP1Δ6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOP1Δ6 is suppressed in either configuration.     

Cloning of the protective antigen gene of Bacillus anthracis

The tripartite protein toxin of Bacillus anthracis consists of protective antigen (PA), edema factor (EF), and lethal factor (LF). As a first step in developing a more efficacious anthrax vaccine, recombinant plasmids containing the PA gene have been isolated. A library was constructed in the E. coli vector pBR322 from Bam HI-generated fragments of the anthrax plasmid, pBA1. Two clones producing PA were identified by screening lysates with ELISA (enzyme-linked immunosorbent assay). Western blots revealed a full-size PA protein in the recombinant E. coli, and a cell elongation assay demonstrated biological activity. Both positive clones had a 6 kb insert of DNA, which mapped in the Bam HI site of the vector. The two inserts are the same except that they lie in opposite orientations with respect to the vector. Thus PA is encoded by the plasmid pBA1.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

Characterization of two Bacillus thuringiensis plasmids whose replication is thermosensitive in B. subtilis

Abstract Two cryptic plasmids of 8.6 and 15 kb, originating from Bacillus thuringiensis , have been cloned in Escherichia coli . The determination of their physical map shows that the 8.6-kb plasmid harbors the transposon Tn 4430 and that the 15-kb plasmid carries Tn 4430 plus one copy of the IS 231 element. The replication regions were identified on the restriction maps and the segregational stability of derived plasmids containing these regions was analyzed in B. subtillis . The results indicate that the stability of these plasmids is negatively correlated to the temperature. After 30 generations, without selective pressure at 51°C, the two types of plasmids are lost.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa

Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.     

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.     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

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.     

Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.