In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.     

Transposition of Tn4560 of Streptomyces fradiae in Mycobacterium smegmatis

Tn4560 (8.6 kb) was derived from Tn4556, a Tn3-like element from Streptomyces fradiae. It contains a viomycin resistance gene that has not been used previously for selection in mycobacteria. Tn4560, cloned in a Streptomyces plasmid, was introduced by electroporation into Mycobacterium smegmatis mc(2)155. Tn4560 transposed into the host genome: there was no obvious target sequence preference, and insertions were in or near several conserved open reading frames. The insertions were located far apart on different AseI macrorestriction fragments. Unexpectedly, the transposon delivery plasmid, pUC1169, derived from the Streptomyces multicopy plasmid pIJ101, replicated partially in M. smegmatis, but was lost spontaneously during subculture. Replication of pUC1169 probably contributed to the relatively high efficiency of Tn4560 delivery: up to 28% of the potential M. smegmatis transformants acquired a stable transposon insertion. The data indicated that Tn4560 may be useful for random mutagenesis of M. smegmatis.     

Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.     

Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis

New derivatives of the tetracycline-resistance transposon Tn1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful for physical mapping, for the generation of new EcoRI sites in cloning experiments, for end-labelling and for sequencing of DNA adjacent to an insertion.     

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).     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

A system for in vivo selection of genomic rearrangements with predetermined endpoints in Escherichia coli using modified Tn10 transposons

Using recombinant DNA techniques, the Tn10-specific tetA gene (coding for tetracycline resistance) has been mutagenized by insertion of a streptomycin-resistance or a kanamycin-resistance gene. The insertions occurred at loci separated by 920 bp. The mutated tetA fragments, respectively designated as Tes (for tetracycline-streptomycin) and Tek (for tetracycline-kanamycin), were subsequently cloned into a phage lambda cIII+cIts857cII+ in replacement of the att lambda region. The two recombinant phages are convenient delivery vehicles which permit the in vivo substitution of the tetA locus of any Tn10 insertion with the Tes or the Tek fragment. The procedure involves two selectable steps: (i) integration of a lambda-Tes (or lambda-Tek) prophage into the Tn10 of interest; (ii) excision of the prophage by a second exchange which leaves the extra resistance gene installed within the Tn10. A major interest of the system is that, once a bacterium carries both Tn10-Tes and Tn10-Tek insertions, a recombination event between the two Tn10 sequences can reconstitute an active tetA gene. This selectable event may be associated with the rearrangement of the sequences surrounding the transposons. This unique property of the "Tes and Tek" system makes it very useful for selection of genomic rearrangements using the Tn10-Tes and Tn10-Tek as predetermined endpoints. The successful isolation of a chromosomal inversion is reported.     

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.     

Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.     

In vivo Himar1-based transposon mutagenesis of Francisella tularensis

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.     

Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe non-viral DNA delivery vehicles

DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process.     

Gene replacement in mycobacteria by using incompatible plasmids

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.     

Highly preferred site of insertion of Tn7 into the chromosome of Vibrio anguillarum.

The possible usefulness of Tn7 as a tool for genetic studies in Vibrio anguillarum was examined. Using the plasmid pRK2073 as the transposon donor, Tn7 transposes at high frequency into the chromosome of V. anguillarum. However, hybridization analysis of the mutants DNA digested with different enzymes revealed that all isolates have the insertions in the same site. This indicates that like in many other gram-negative bacteria, Tn7 shows a specificity of transposition in the chromosome of V. anguillarum. Plasmid pRK2013 proved to be a very useful delivery vector for transposon mutagenesis in V. anguillarum.     

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis.

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.     

Use of phenotypic suppression for direct selection of loss of aminoglycoside antibiotic resistance determinants

Summary A strain of Escherichia coli containing a conditional drug dependent arginine auxotrophy was used to select for the loss of plasmid and/or transposon encoded kanamycin (Km) or streptomycin (Sm) resistance determinants. Because these determinants inactivate the corresponding drug thus elminating drug suppression, loss of the drug-resistance determinants was selected directly by growth on minimal media plates containing sub-lethal dosages of the drug. This method was used to select loss of Km or Sm resistance determinants due to loss of plasmids, transposition from plasmid to chromosome, and eduction of transposons from the chromosome. Drug suppression was compared to phage PRD1 resistance in selecting for loss of plasmid vehicles during transposition and was found to be 10-1,000 times more efficient. Eighty percent of the eductant clones had undergone imprecise eductions suggesting that this method may be useful in selecting stable deletion mutants. An antibiotic suppressible strain of Pseudomonas stutzeri was obtained implying a broad utility of this selection procedure.     

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.     

Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as mucosal delivery vehicles for vaccinal, medical or technological use has been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins and for plasmid DNA delivery to eukaryotic cells. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) and more recently to efficiently transfer DNA to eukaryotic cells. A promising application of L. lactis is its use for the development of live mucosal vaccines. Here, we have reviewed the expression of heterologous protein and the various delivery systems developed for L. lactis, as well as its use as an oral vaccine carrier.     

Rapid construction of sequencing templates by random insertion of antibiotic resistance genes

We describe a novel and handy method for generating a population of templates for sequencing. The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I. The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid. DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background. We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants.