Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites

Abstract We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene ( tsr ) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces ( tsr ) and E. coli (β-lactamase ( bla ) and β-galactosidase ( lacZ )). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans , and are compatible with pIJ702.     

Construction and intergeneric conjugative transfer of a pSG5-based cosmid vector from Escherichia coli to the polyisoprene rubber degrading strain Micromonospora aurantiaca W2b

A non-rubber degrading mutant of the polyisoprene rubber degrading bacterium Micromonospora aurantiaca W2b lacking the capability to form halos on latex overlay agar plates was isolated after N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. A 10.3-kb shuttle cosmid vector pGM446 was constructed from the Streptomyces cloning vectors pGM160 and pOJ446. This vector was transferred by conjugation from Escherichia coli to M. aurantiaca W2b. The frequency of formation of exconjugants with pGM446 was 3.6 x 10(-3). This vector could be useful for shotgun cloning of genes into the non-rubber degrading mutant L1 from M. aurantiaca W2b.     

Construction of a shuttle lacmZα-based Escherichia coli-actinomycetes vector containing the phage JHJ-3 replicon

Using the broad replicating range JHJ-3 phage replicon, a shuttle vector for Escherichia coli and actinomycetes has been constructed. The vector, pOJ31, bears the lacZ alpha fragment allowing a blue/white gene cloning system. pOJ31 also contains a polylinker of 15 unique cloning sites and the phage T7 promoter. The vector has been used to stably express the mel gene from plasmid pIJ702 in Streptomyces lividans.     

pUCS75, a stable high-copy-number Streptomyces—Escherichia coli shuttle vector which facilitates subcloning from pUC plasmid and M 13 phage vectors

A new Streptomyces-Escherichia coli shuttle vector, pUCS75, has been constructed to permit facile subcloning of DNA from the multiple cloning sites of the pUC plasmid and M13 phage vectors. In contrast to other commonly used shuttle vectors, pUCS75 retains the primary site for second-strand synthesis (ssi) of the parental streptomycete replicon, pIJ101. This sequence can not only enhance structural stability of the plasmid, but also confers on it an elevated copy number when replicated in Streptomyces. Consequently, the vector is useful for cloning sequences containing repeat structures and for allowing the high-level expression of cloned genes.     

Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells.

In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ' sequence, carried by our vectors.     

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.     

Shuttle vectors for hyperthermophilic archaea

Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea. Received: January 12, 1997 / Accepted: May 29, 1997     

A Host–Vector System for a Cellulose-Producing Acetobacter Strain

An indigenous plasmid, named pAH4, was detected in a cellulose-producing Acetobacter strain. This plasmid, consisting of 4002 bp, contained an AT-rich region and encoded several open reading frames, as deduced by the complete nucleotide sequence. One of the putative open reading frames showed homology with replication proteins of other plasmids. A shuttle vector of Escherichia coli and this strain was constructed by connecting pAH4 to pUC18. Electroporation of the shuttle vector into the strain yielded 1.7 × 10⁵ ampicillin resistant transformants per μg DNA. The shuttle plasmid was very stably maintained in the strain.     

Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

Mechanism of the Formation of γ-Glutamylpeptides in Glutamic Acid Fermentations

We have constructed phage cloning vectors from an actinophage, R4. A deletion derivative (R4 Δ22B) which had a BamHI linker inserted at the unique PvuII site was used to clone the thiostreptone resistant (tsr) gene derived from plasmid vector pIJ365. The tsr derivative obtained, R4Δ22B-tsr1, was shown to have the same level of thiostreptone resistance in lysogenized cells as that of pIJ365-carrying cells. Under the optimal conditions, R4Δ22B-tsr1 phage was lysogenized at a frequency of 5x10^-2 per infected phage. The usefulness of R4 phage derivatives for gene cloning is discussed.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Summary Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit lethal zygosis and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Versatile shuttle cloning vectors for the unicellular cyanobacterium Anacystis nidulans R2

Abstract 3 new shuttle cloning vectors for gene transfer into Escherichia coli and Anacystis nidulans have been constructed by utilizing the cyanobacterial origin of replication of the small plasmid pANS from A. nidulans . 2 of these new vectors, pXB7 (pDPL13 derivative) and pECAN8 (pUC8 derivative), convey ampicillin resistance, and transform A. nidulans with relatively high frequencies. Vector pXB7 has 10 unique cloning sites; pECAN8 contains 4 cloning sites within the lacZ gene permitting rapid detection of DNA inserts in the presence of Xgal. The third vector, pKBX, has a lower transformation frequency but adds kanamycin resistance as a selectable gene for shuttle vectors of cyanobacteria.     

Transformation ofStreptomyces granaticolor with natural and recombinant plasmid vectors

Protoplasts of Streptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350--pBR322) isolated from E. coli cells were used. The onset of blue colour granaticin production by S. granaticolor cells was used as a marker to prepare protoplasts with a high transformation capacity. The presence of a restriction system is discussed.     

Sequencing and analysis of three plasmids from <Emphasis Type="Italic">Lactobacillus casei</Emphasis> TISTR1341 and development of plasmid-derived <Emphasis Type="Italic">Escherichia coli–L. casei</Emphasis> shuttle vectors

Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.     

The Use of Epstein–Barr Virus-Based Shuttle Vectors in Rat PC12 Cells

1. The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death. Thus the abilityto transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells.2. We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells.3. The EBV-based vectors replicate episomally in PC12 cells for at least 2months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes.3. PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably.4. Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells.5. The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells.     

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.     

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.     

A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.

     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.     

Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector

The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E. coli positive selection shuttle vector. The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E. coli and Streptomyces lividans respectively. The positive selection shuttle vector pLR591 facilitates the construction in E. coli of genomic libraries which can be screened in Streptomyces strains.