Broad host range cloning vectors for gram-negative bacteria

A series of cloning vectors has been constructed based on the broad-host-range plasmid R300B. One of these vectors, pGSS33, has a size of 13.4 kb and carries four antibiotic resistance genes [ampicillin (Apr), chloramphenicol (Cmr), streptomycin (Smr) and tetracycline (Tcr)], all of which have restriction sites for insertional inactivation. The derivation, structure and uses of the plasmids are described.     

Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).     

Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria

Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.     

Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis

Summary A set of vectors was constructed for the cloning and expression of heterologous genes in the Gramnegative bacterium Zymomonas mobilis under the control of the pdc promoter of Z. mobilis. The vectors pPTZ1, pPTZ3, and pPTZ4 are based on the cryptic Z. mobilis plasmid pZM02 and on parts of the Escherichia coli plasmids pKK223-3 and pBR322 together with the multiple cloning site of phage Ml3mp18. DNA fragments can be readily inserted immediately downstream from the pdc promoter at unique restriction sites for KpnI, XbaI and PstI in pPTZl and additionally for SmaI and BamHI in pPTZ3. In pPTZ4, the 5 terminal codons of pdc were deleted allowing the formation of gene fusions. Expression of a promoterless chloramphenicol acetyltransferase gene (cat) controlled by the pdc gene promoter resulted in enzyme activities of up to 5.5 U/mg total cell protein in Z. mobilis cells.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

Broad-host-range mobilizable suicide vectors for promoter trapping in gram-negative bacteria

Here we report the construction of three different vectors for the identification of bacterial genes induced in vitro and/or in vivo. These plasmids contain kanamycin, gentamicin, or tetracycline resistance genes as selectable markers. A promoterless cat and an improved GFP (mut3-gfp) can be used to follow the induction of gene expression by measuring chloramphenicol resistance and fluorescence, respectively.     

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

Background  

The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.     

Adeno-cosmid cloning vectors for regulated gene expression

Background

Adenovectors are widely used for efficient delivery of genes into a variety of cell types and organisms. However, the construction of the desired vector/genes combination, especially if it involves the cloning of several gene cassettes, can be laborious due to the large size of these vectors. New methods are needed to simplify the construction of complex combinations of gene cassettes into adenovectors.

Methods

Using simple cloning techniques and exploiting the λ‐phage packaging system, we devised efficient methods for the ‘selection’ of the desired vector constructs. Thus we generated a series of cosmids containing the adeno helper dependent (HD) backbone in which we inserted cis‐ and trans‐acting tetracycline (tet) elements for the regulation of any gene of interest. One of these cosmids has been used to produce an HD adenovirus carrying a tetracycline‐regulated gene expressing β‐galactosidase.

Results

We have demonstrated that the adeno‐cosmid system allows rapid and efficient cloning of genes of interest in helper dependent vectors, and described a prototype ‘ready‐to‐use’ vector in which any gene of interest can be easily expressed under the control of the tet system. The HD viruses produced with this novel methodology can be grown at high titers, can be easily separated from the helper adenovirus, and allow delivery and regulated gene expression in a variety of tissues.

Conclusions

Exploiting the λ‐packaging system, complex adeno constructs can be generated with a simple and reproducible protocol, which allows selection of the desired size construct, counterselecting for the frequently observed intramolecular recombinations and deletions. Copyright © 2002 John Wiley & Sons, Ltd.

     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Construction of a mobilizable cloning vector for site-directed mutagenesis of gram-negative bacteria: application to Rhizobium leguminosarum

A mobilizable cloning vector was constructed from defined fragments to serve as a suicide plasmid for site-directed mutagenesis. The new vector, pKOK4, closely resembles plasmid pBR325. However, the inverted duplication existing in the latter was not introduced. The useful cloning sites of pBR325 (EcoRI, HindIII, EcoRV, BamHI, SalI, PstI and PvuI) were retained and are located in one of the three resistance markers, ApR, CmR or TcR, respectively. Also, in pKOK4 the CmR gene retains its own promoter. The mob site of plasmid RP4 was introduced as a 760-bp fragment at a defined location. The mobilization frequency of pKOK4 within Escherichia coli strains is approx. 4 x 10(-2) per recipient cell. The size of pKOK4, deduced from the construction, is 6368 bp. We used pKOK4 for site-directed mutagenesis of hup-specific DNA from Rhizobium leguminosarum B10. Integration of the vector could be distinguished reliably from marker exchange by screening for the antibiotic resistance(s) of the plasmid. This reduced the number of clones to be retested by colony and Southern hybridization to approx. 1% of the original number. Of these, almost 70% contained the desired marker exchange.