Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1

Summary The plasmids R15 and RP4:: Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA ^– Escherichia coli cells and have a mean life-time of more than 50 generations in a recA ⁺ background.The 85 Md cointegrates were generated at a frequency of 4×10^–4 per R15 transconjugant during a mating between E. coli [R15; RP4:: Tn1] and E. coli [FColVBtrp:: Tn1755]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4:: Tn1. The transposition of IS8 from RP4:: Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of FColVBtrp:: Tn1755 into E. coli [R15; RP4:: Tn1] cells.The formation of the 92 Md cointegrates occurs at a frequency of 2×10^–5. The fused molecules of R15 and RP4:: Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, BglII and SalI and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.Abbreviations Ap ampicillin - EtBr ethidium bromide - Km kanamycin - Md megadaltons - Sm streptomycin - Su sulfanilamide - Tc tetracycline - [] brackets indicate plasmid-carrier state     

Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards

The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc^r) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc^r determinants. Tc^r was determined to be due to the presence of Tet B in Pantoea agglomerans and other members of the family Enterobacteriacae and Tet A, Tet C, or Tet G in most Pseudomonas isolates. The cause of Tc^r was not identified in 16% of the isolates studied. The Tc^r genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn5393. Transposable elements with Tc^r determinants were detected by entrapment following introduction into Escherichia coli. Tet B was found within Tn10. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn10; one had IS911 located within IS10-R and one had Tn1000 located upstream of Tet B. Tet A was found within a novel variant of Tn1721, named Tn1720, which lacks the left-end orfI of Tn1721. Tet C was located within a 19-kb transposon, Tn1404, with transposition genes similar to those of Tn501, streptomycin (aadA2) and sulfonamide (sulI) resistance genes within an integron, Tet C flanked by direct repeats of IS26, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs^r) cassette linked to a bacterial tetracycline resistance (tet^r) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs^r-tet^r cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.     

Genetic analysis of Tn7 transposition

Summary The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7. Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites. The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined. All the deleted Tn7s isolated in this work had lost their transposing capability. The possibility of complementing them was studied using plasmids containing all or part of Tn7. Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function. Four other deleted Tn7s could be complemented by Tn7. One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition. The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb. Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7. The classification of Tn7 with regard to the other transposable elements is discussed.     

High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon

Summary A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventrional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn5501 and Tn5502, are cryptic transposons of the Tn3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon.     

Transposon Mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: Physical Analysis of Tn5 and Tn5-Mob Insertion Mutants

Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10⁻⁷ per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km^r Sm^rA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.     

Map of plasmid RP4 derived by insertion of transposon C.

We have determined the location of 36 sites on plasmid RP4 into which transposon C (an 8.5 × 10⁶ molecular weight DNA sequence conferring trimethoprim and streptomycin resistance) had spontaneously inserted itself. These were located by sucrose gradient analysis of EcoRI-generated and then, separately, the HindIII-generated DNA fragments from each RP4-TnC² plasmid. RP4 has a single EcoRI-susceptible site and, suitably displaced from this, a HindIII-susceptible site, whereas TnC has, respectively, one and two sites for these two enzymes. Thus the sizes of the restriction fragments depend on the location and orientation of the inserted TnC.Some of the RP4-TnC clones had lost one of the RP4 characters: transferability (Tra), tetracycline (Tc) or kanamycin (Km) resistances, but no ampicillin (Ap) sensitive clones were detected. Insertions giving each of these phenotypic changes cluster together at positions on the circular RP4 map that presumably locate the genes responsible for the Tra⁺, Km^r and Tc^r phenotypes. The Tra^? plasmids were grouped into four classes on the basis of their conferred phage sensitivities and plasmid copy numbers. The gene giving Ap^r was located by its known proximity to a BamHI-susceptible site. All the plasmids analysed had TnC inserted with one particular orientation. TnC insertions giving no detectable phenotypic change were not randomly placed around RP4, but clustered into certain regions. Two large regions, one containing TnA, had no TnC insertions. Ligation experiments with restriction fragments from various RP4-TnC plasmids led to the conclusion that both these regions contain genes essential to the replication and maintenance of RP4. The location of the HindIII site of RP4 within the gene giving Km^r should prove valuable to the use of this plasmid as a cloning vehicle.     

Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv. tomato

Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Km^r transconjugants occurred at frequencies ranging from 2 × 10⁻⁷ to 9 × 10⁻⁶; approximately 5.5% of these transconjugants were also Cm^r, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km^r Cm^s mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Km^r Cm^s transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Km^r Cm^s transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.     

A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant

This paper describes a simple method for the isolation of small plasmids of various sizes from pSMI, a derivative of the resistance plasmid R 100. The method is based on the observation that a repressor-negative mutant of the ampicillin-resistance (amp^r) transposon Tn3, Tn3 No. 5, mediates cointegration of a plasmid carrying Tn 3 No. 5 (pMB8::Tn 3 No. 5) into virtually any site on pSMI. The resulting cointegrate plasmids contain the pSMI sequence which is joined with the amp^r gene of the Tn 3 mutant. This cointegration is so frequent that large cointegrate plasmids can be readily detected in the total plasmid DNA prepared from cells carrying pSMI and pMB8::Tn3 No. 5. We were able to isolate small plasmids of various sizes by digesting the total plasmid DNAs with restriction endonucleases which cut both pSM 1 and Tn3 No. 5 sequences present in the cointegrates and subsequently ligating the restriction fragment containing both the amp^r gene and the region necessary for replication of pSMI. Analysis of these plasmids, named pBV plasmids, with restriction endonucleases and by nucleotide sequencing allowed us to determine regions necessary or unnecessary for replication, thus defining a minimal replication region of pSMI. The present method is generally useful for the isolation of small derivatives from any large plasmid for the study of genes and sites adjacent to or within the minimal replication region of the plasmid.     

Control of Tn7 transposition

Summary Our isolate of Tn7 (named Tn7S) contains an IS1 insertion, and this IS1 can be converted into Tn9. In vitro and in vivo deletions of Tn7S and Tn7S:: Tn9 define regions of the transposon required for antibiotic resistance and transposition. Complementation of deletion mutants by cloned Tn7 fragments indicates the existence of two regions, denoted tnp7A and tnp7B, required for all transposition events. Another region, denoted tnp7C, is required for transposition from the chromosome to RP1 but not for transposition from a small IncP-1 replicon to the chromosome. The presence of Tn7S terminal sequences in an RP1 replicon reduces the transposition of a second Tn7S derivative from the chromosome by about one order of magnitude. The measured frequency of Tn7S transpositions from a small IncP-1 replicon to the chromosome depends on the particular incompatibility system used to eliminate that replicon. Genetic and physical data indicate that high frequencies of Tn7S transposition to the chromosome (40%) are triggered by the IncP-1 incompatibility reaction, thus suggesting the existence of a Tn7 mechanism for sensing the state of the carrier replicon.     

Genetic Construction of Lactose-Utilizing Xanthomonas campestris

Xanthomonas campestris, the producer of xanthan gum, possesses a β-galactosidase of very low specific activity. Plasmid pGC9114 (RP1::Tn951), generated by the transposition of the lactose transposon Tn951 to RP1, was conjugally transferred into XN1, a nalidixic acid-resistant derivative of X. campestris NRRL B-1459S-4L. Transfer occurred on membrane filters and in broth. The β-galactosidase gene of Tn951 was expressed in X. campestris. The specific activity of β-galactosidase in transconjugants was over 200-fold higher than that in XN1, and transconjugants grew as well in lactose-based media as in glucose-based media. The lactose-utilizing transconjugants could potentially be used to produce xanthan gum from cheese whey.     

Specificity of Transposon Tn5 Insertion

Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.     

Sequence of the 68,869 bp IncP-1α plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements

To analyse the significance of conjugative broad-host-range IncP-1α plasmids for the spread of antibiotic resistance determinants in waste-water treatment plants we isolated and characterised five different IncP-1α plasmids from bacteria of activated sludge and the final effluents of a municipal waste-water treatment plant. These plasmids mediate resistance to ampicillin, cefaclor, cefuroxime, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, tobramycin, and trimethoprim. The complete 68,869 bp DNA-sequence of the IncP-1α plasmid pTB11 was determined. The pTB11 backbone modules for replication (Rep), mating pair formation (Trb), multimer resolution (Mrs), post-segregational killing (Psk), conjugative DNA-transfer (Tra), plasmid control (Ctl), and stable maintenance and inheritance (KilA, KilE, and KilC) are highly conserved as compared to the ‘Birmingham’ IncP-1α plasmids. In contrast to the ‘Birmingham’ plasmids pTB11 carries an insert of a Tn402-derivative integrating a class 1 integron in the intergenic region between the multimer resolution operon parCBA and the post-segregational killing operon parDE. The integron comprises the resistance gene cassettes oxa2 (β-lactamase), aacA4 (aminoglycoside-6′N-acetyltransferase), and aadA1 (aminoglycoside-3′-adenylyltransferase) and a complete tniABQR transposition module. Integron-specific sequences were also identified on other IncP-1α plasmids analysed in this work. In contrast to the ‘Birmingham’ plasmids the pTB11 tetracycline resistance module carries a pecM- and a pncA-like gene downstream of the tetracycline resistance gene tetA and contains an insertion of the new insertion sequence element ISTB11. The transposable elements IS21 and Tn1 which disrupted, respectively, orf7 and klcB on the ‘Birmingham’ plasmids are not present on pTB11. Identification of IncP-1α plasmids in bacteria of the waste-water treatment plant’s final effluents indicates that bacteria carrying these kind of plasmids are released into the environment.     

Tn7-encoded proteins

Summary Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1:: Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the lefthand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.     

Cloning of a lac+, bamHI fragment into transposon Tn3 and transposition of the Tn3[lac] element

By use of recombinant DNA techniques, we have inserted the lac⁺ operon into a transposon (Tn3). We constructed the recombinant in such a way that the essential step in assaying for transposition consisted of screening for bacteria with a thermostable Lac⁺ phenotype. Our results showed that transposition of the Tn3[lac⁺] element occurred and that its frequency was derepressed compared to frequencies reported by others for wild-type Tn3 transposition.     

Transposon Tn21 encodes a RecA-independent site-specific integration system

Summary The IncW plasmid R388 and the DNA region of Tn21 containing the Sm^r and the Su^r genes are capable of RecA-independent recombination. This recombination occurs at a relatively high frequency (up to 10^-4 recombinants per recipient molecule) and results in integration of the two plasmids. No detectable repeats are formed in the process. The crossover points have been confined to a 0.4-kb homologous segment in both plasmids which contains a 59-bp DNA sequence presumably involved in the acquisition of new genes by Tn21 and its relatives (Cameron et al. 1986). It is likely that the recombination occurs precisely at this point. At least one trans-acting function (an integrase) is required for the site-specific recombination. It has been localized to a 1456-bp BstEII-BamHI fragment of Tn21 and can efficiently complement the integration of plasmids containing the integration site.