Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.     

Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.     

A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance.

Summary This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, BglI, BglII, HindII, HindIII, HpaI, SalI, AvaI, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColE1::Tn5 plasmids, and a ColE1::Tn5 deletion derivative. BalI, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColE1::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a BglII site within this segment results in loss of the neomycin resistance phenotype. Since this BglII site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.     

Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance.

A cleavage map of a recombinant plasmid carrying Tn10 was constructed for 13 different restriction enzymes. The Tn10 region of this plasmid contains cleavage sites for BamHI, AvaI, BglI, BglII, EcoRI, XbaI, HincII, HindIII, and HpaI. Restriction enzymes PstI, SmaI, KpnI, XhoI, SalI, and PvuI do not cleave within the Tn10 element. This map confirms the previously reported structure of this transposon; it is composed of a unique sequence (approximately6,400 base pairs long), which in part codes for the tetracycline resistance functions and is bounded by inverted repeats (approximately 1,450 base pairs long).     

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Plasmid pAS8Tc^s rep-1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas. The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

Tn292l, a transposon encoding fosfomycin resistance.

The determinant of resistance to fosfomycin of the Serratia marcescens plasmid pOU900 was transposed into the plasmid ColE1 and into the plasmid RP4 in the absence of the RecA function of the host. In each case, the acquisition of fosfomycin resistance was correlated with the presence of a discrete piece of DNA, uniform in size and in restriction pattern, This new, 7.5-megadalton transposable element encoding resistance to fosfomycin has been called Tn2921. A preliminary map of the transposon is presented.     

Denaturation map of the ColE1-Km plasmid pCR11.

The denaturation map of EcoRI-digested pCR11, a ColE1-Km plasmid, is described. The 2.0 kilobase ColE1-derived segment contains an adenine+thymine rich site in the colicin immunity gene region. In the 7.2 kilobase kanamycin resistance region, the transposon Tn903 consists of an adenine+thymine rich 0.98 kilobase kan gene region flanked by a guanine+cytosine rich 1.09 kilobase inverted duplication.     

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.     

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.     

Properties of transposon Tn2555 carrying the genes for sucrose utilization

Tn2555, a new transposon coding for genes of sucrose utilization was studied. Tn2555 was shown to integrate into the plasmids RP4 and R6K, phage P1CmClr100 and Escherichia coli K12 chromosome. Tn2555 frequency of transposition to RP4 and R6K DNA is (2-5) X 10(-7) in Rec+-strain, (3-6) X 10(-8) in Rec--strain. Tn2555 integration site in phage P1CmClr100 Sac+-derivative studied has been localised within the C-segment of P1 DNA. In three independent cases of Tn2555 transposition to the chromosome the transposon was found to be integrated in the region between 29 and 32 min of Escherichia coli K12 linkage map. The restriction endonuclease analysis of seven independent isolates of RP4::Tn2555 has shown the grouping of Tn2555 integration sites in the Tn1 region of RP4. Frequent rearrangements occurring within Tn2555 have been revealed by the analysis. However, an invertible DNA segment of about 6-7 kb was preserved in all transposon structures.     

Tn4527, a Tp Sp/Sm transposon related to Tn7 and flanked by IS1

Tn4527 was isolated from a Salmonella typhimurium strain obtained in Brazil. Its size is 19.6 kb and it carries resistance to trimethoprim, spectinomycin, and streptomycin, as in the case of Tn7 (14 kb). A restriction analysis of the transposon shows regions of similarity to Tn7 mixed with extra DNA. The 2.6-kb and 2.2-kb HindIII fragments of Tn7, which encode transposition-related proteins, show homology to Tn4527. In contrast to Tn7, Tn4527 is flanked by direct repeats, which seem to be IS1's, as they have appropriate restriction sites and hybridize both to IS1 and to internal IS1 oligonucleotides.     

Physical and functional mapping of Tn2603, a transposon encoding ampicillin, streptomycin, sulfonamide, and mercury resistance

Summary A map of cleavage sites for restriction endonucleases EcoR1, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.:: Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.     

Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid.

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.     

Characteristics of Tn3611 and Tn3613 transposons discovered in the chromosome of the BS205 strain of Pseudomonas aeruginosa

The transposons Tn3611 (HgR, 9.0 kb) and Tn3613 (SmRCmRKmRSuRHgR, 24.0 kb) were discovered in the chromosome of Pseudomonas aeruginosa BS205. The physical and genetic maps of these transposons were constructed using restriction endonucleases EcoRI, BamHI, SalI, HindIII. Genes of the mer operon, tnpA and tnpR were localized in the Tn3611 transposon, the genes DHPS-II, aad, tnpA, tnpR being situated in the map of the Tn3613 transposon. It was established that the Tn3611 belongs to the class of Tn3-like transposons and the Tn3613 is a complex transposon flanked by inverted repeats 2.2 kb in size, the transposon comprising both Tn3611 and a migrating sequence.