PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria

We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P_BAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.     

Construction of improved vectors for protein production in Pseudomonas aeruginosa

We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Saccharomyces cerevisiae-Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.     

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.     

Resistance to Tellurite as a Selection Marker for Genetic Manipulations of Pseudomonas Strains

Resistance to the toxic compound potassium tellurite (Tel^r) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release. In this study, the activated Tel^r determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors. In one case, the Tel^r determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons. In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets. Either in the plasmid or in the transposon vector, the Tel^r marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes. This allowed construction of antibiotic resistance-free but selectable P. putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway.     

Development of a Gateway-compatible two-component expression vector system for plants

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

     

Construction of new cloning vectors that employ the phytoene synthase encoding gene for color screening of cloned DNA inserts in Thermus thermophilus

Strains of Thermus thermophilus produce unique carotenoids called thermozeaxanthins and their colonies are light-yellow pigmented. Here, we developed a new cloning system allowing for the rapid and convenient detection of recombinants by color screening based on carotenoid production in T. thermophilus. We constructed two cloning vectors that overexpress the crtB gene encoding a phytoene synthase under the strong promoter of the slpA gene. Phytoene synthase is one of essential enzymes for the production of carotenoids. We also isolated a carotenoid-overproducing mutant that formed orange colonies. Because disruption of crtB in the carotenoid-overproducing mutant resulted in white colonies, we used the disruptant as a host strain. Whereas transformants carrying a new cloning vector, pTRK1-PRslpA-crtBcas, grew into unusual red-pigmented colonies probably because of the extreme accumulation of thermozeaxanthins, those carrying the vector with a foreign DNA inserts formed white colonies. Thus, recombinants can be detected easily by color screening (red/white screening) in T. thermophilus. This cloning system requires no additional chromogenic substrate in the medium. We also constructed a promoter-probe vector, pTRK1-crtBmcs-PP, employing the open reading frame of crtB with multiple cloning sites. Using this vector, a series of colony-color phenotypes is observed probably depending on promoter activities of foreign DNA inserts, which enables the rapid probing of promoters. These vectors are useful to simplify cloning procedures and to identify the promoters of different strengths in T. thermophilus.     

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.