Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads

Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).     

IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads

Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7). A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid. Transposition of IS50L-tox into the chromosome of P. fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element. A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase. The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species. Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).     

Survival of genetically engineered Pseudomonas fluorescens in soil in competition with the parent strain

Abstract The population dynamics of two genetically engineered Pseudomonas fluorescens strains, D5 and C5t, introduced into a loamy sand soil, in competition with a spontaneous antibiotic-resistant mutant of the corresponding wildtype strain was studied. Strain D5 contained an insertion of transposon Tn5 in its genome, whereas strain C5t was obtained by insertion of Tn 5 :: tox , a Tn 5 -derivative containing a Bacillus thuringiensis var. morrisoni δ-endotoxin gene, into the chromosome using a suicide vector system. Southern hybridization analysis demonstrated the absence of vector sequences, and the presence of single copies of either Tn 5 or Tn 5 :: tox in the respective strains. Western blotting and a bio-assay on larvae of Anopheles stephensi suggested the tox gene was functional in clone C5t. Both D5 and C5t were prototrophic and their generation times in minimal medium were slightly below that of the corresponding wild-type strain. Tn 5 and Tn 5 :: tox were stable in both clones during growth in minimal medium for 16 generations. During growth in competition with the wild-type strain, D5 competed well, however C5t was outcompeted from 50 to below 3% of the population in 40 generations. During growth in competition in the sterile loamy sand, both strains were outcompeted by the parent strain; strain C5t was less competitive than D5. In non-sterile loamy sand, the introduced mixed populations showed a slow decline; both C5t and D5 were outcompeted by the parent strain. The decreased fitness of both modified strains, although significant, was considered to be small in ecological terms. Further, the addition of 10% bentonite clay to the loamy sand resulted in a significant enhancement of survival of the mixed populations, and a stabilization of the proportions between the modified strains and the parent. Finally, there was a trend towards a decrease in the proportion modified strain/parent strain in both mixes in the rhizosphere of wheat.     

Cloning and orientation of the gene encoding polynucleotide phosphorylase in Escherichia coli.

Mutations which affect the activity of polynucleotide phosphorylase (PNPase) map near 69 min on the bacterial chromosome. This region of the chromosome has been cloned by inserting the kanamycin-resistant transposon Tn5 near the argG and mtr loci at 68.5 min. Large SalI fragments of chromosomal DNA containing the Tn5 element were inserted into pBR322, and selection was made for kanamycin-resistant recombinant plasmids. Two of these plasmids were found to produce high levels of PNPase activity in both wild-type and host strains lacking PNPase activity. The pnp gene was further localized and subcloned on a 4.8 kilobase HindIII-EcoRI fragment. This fragment was shown to encode an 84,000-molecular weight protein which comigrated with purified PNPase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The orientation of the pnp gene was determined by insertion of Tn5 into the 4.8 kilobase fragment cloned in pBR322. Some of the insertions had lost the ability to elevate the level of PNPase activity in the host bacterium. Restriction mapping of the positions of the Tn5 insertions and analysis of plasmid-encoded polypeptides in UV-irradiated maxi-cells indicated that the pnp gene is oriented in the counterclockwise direction on the bacterial chromosome.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Orientation of the tox gene in the prophage of corynebacteriophage beta.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRM- has also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.     

Construction of transposons carrying the transfer functions of RP4

The transfer genes and origin of transfer of the wide host range plasmid RP4 have been cloned into the transposons Tn1 and Tn5. The newly constructed transposons can be used to mutagenize bacterial plasmids or the chromosome in species such as Escherichia coli or Rhizobium. It is then possible to mobilize the plasmid or chromosome using the transfer functions provided in cis by the transposon. These constructs may aid chromosome mapping in many gram-negative species by allowing the wider use of the RP4 conjugal transfer system combined with the potential ability to select the site of insertion and thus the site of the origin of transfer.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Conferral of transposable properties to a chromosomal gene in Escherichia coli

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK⁺-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.     

Transposon-facilitated recombination in Vibrio cholerae

Summary Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide portable regions of homology. The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P:Tn1 plasmids that contained Tn1 inserted in the opposite orientation.     

Use of transposons for studying the genetic organization of Vibrio cholerae non 01]

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.     

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.     

Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5.

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell. The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.     

Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.