Transformation of Mycobacterium smegmatis with Escherichia coii plasmids carrying a selectable resistance marker

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.     

A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation

Agrobacterium rhizogenes, along with A. tumefaciens, has been used to affect genetic transformation in plants for many years. Detailed studies conducted in the past have uncovered the basic mechanism of foreign gene transfer and the implication of Ri/Ti plasmids in this process. A number of reviews exist describing the usage of binary vectors with A. tumefaciens, but no comprehensive account of the numerous binary vectors employed with A. rhizogenes and their successful applications has been published till date. In this review, we recollect a brief history of development of Ri-plasmid/Ri-T-DNA based binary vectors systems and their successful implementation with A. rhizogenes for different applications. The modification of native Ri plasmid to introduce foreign genes followed by development of binary vector using Ri plasmid and how it facilitated rapid and feasible genetic manipulation, earlier impossible with native Ri plasmid, have been discussed. An important milestone was the development of inducible plant expressing promoter systems which made expression of toxic genes in plant systems possible. The successful application of binary vectors in conjunction with A. rhizogenes in gene silencing and genome editing studies which are relatively newer developments, demonstrating the amenability and adaptability of hairy roots systems to make possible studying previously intractable research areas have been summarized in the present review.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.     

An EBV‐based plasmid can replicate and maintain in stem cells

Viral vectors have a wide range of applications in biology, particularly in gene therapy. Based on their integration capacity, viral vectors are classified as either integrating or non‐integrating vectors. Although integrating vectors, such as lentivectors, have the ability to direct prolonged expression of exogenous genes, manipulation of the host genome is an inappropriate feature of these gene delivery tools. Non‐integrating vectors, such as episomal replicating plasmids, can replicate and persist in host cells for long periods without any chromosomal interruption. These advantages made them good tools for gene induction purposes in gene therapy and basic studies. Due to the necessity of gene induction in stem cells for study of mammalian development and targeted differentiation, the use of integrating vectors for prolonged expression of genes of interest has been developed. Application of replicating plasmids can overcome some drawbacks associated with integrating vectors, although replication and maintenance of these plasmids can differ between cell types. Previously, it has been shown that such plasmids can be maintained in human embryonic stem cells for more than one month, but the rate of the plasmid replication during the host cell cycle has not been elucidated. In the present study, we showed that an EBV‐based plasmid can replicate simultaneously with host in pluripotent and multipotent human and mouse stem cells and can be sustained for long time periods in dividing cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1579–1585, 2015     

Development of a microfluidic chip-based plasmid miniprep

Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16 h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10⁵ times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.     

The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110

Gateway^® cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method.     

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a θ-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.     

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.     

New cloning vectors using benomyl resistance as a dominant marker for selection inNeurospora crassa and in other filamentous fungi

We describe five new plasmid vectors derived from pBR322 that carry theNeurospora crassa β-tubulin gene conferring resistance to benomyl. The benomyl resistance gene has been modified to eliminate an internalEcoRI site to facilitate the cloning ofEcoRI restriction fragments. These plasmids allow rapid subcloning of fragments from one replicon to another without insert fragment purification due to the presence of different drug resistance markers (resistance to kanamycin, tetracycline, or chloramphenicol) carried on the plasmids. These vectors will allow rapid transformation ofN. crassa and other filamentous fungi to allow phenotypic characterization of subcloned fragments.     

Elucidation of Insertion Elements Carried on Plasmids and In Vitro Construction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix

Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6Kγ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.     

Genetic and functional properties of the self-transmissible Yersinia enterocolitica plasmid pYE854, which mobilizes the virulence plasmid pYV

Yersinia strains frequently harbor plasmids, of which the virulence plasmid pYV, indigenous in pathogenic strains, has been thoroughly characterized during the last decades. Yet, it has been unknown whether the nonconjugative pYV can be transferred by helper plasmids naturally occurring in this genus. We have isolated the conjugative plasmids pYE854 (95.5 kb) and pYE966 (70 kb) from a nonpathogenic and a pathogenic Yersinia enterocolitica strain, respectively, and demonstrate that both plasmids are able to mobilize pYV. The complete sequence of pYE854 has been determined. The transfer proteins and oriT of the plasmid reveal similarities to the F factor. However, the pYE854 replicon does not belong to the IncF group and is more closely related to a plasmid of gram-positive bacteria. Plasmid pYE966 is very similar to pYE854 but lacks two DNA regions of the larger plasmid that are dispensable for conjugation.     

Deletion of the <Emphasis Type="Italic">Clostridium thermocellum recA</Emphasis> gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥?60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ?recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.     

Development of a New Generation of Vectors for Gene Expression,Gene Replacement,and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.     

Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

Background

Temperature-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996) for predicting Ts mutants based on the amino-acid sequence of the protein.

Results

A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A) was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C.

Conclusions

The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.