Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon.

Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described. This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [rpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.     

Endpoint Bias in Large Tn10-Catalyzed Inversions in Salmonella Typhimurium

A genetic strategy identified Salmonella typhimurium strains carrying large (>40 kb) Tn10-catalyzed inversions; the inverted segments were characterized by XbaI digestion and pulsed field gel electrophoresis. Two size classes of large inversions were found. More than half of the inversions extended 40-80 kb either clockwise or counterclockwise of the original Tn10 site. The remaining inversions extended up to 1620 kb (33% of the genome), but the distal endpoints of these inversions were not randomly scattered throughout the chromosome. Rather, each Tn10 repeatedly yielded similar (though not identical) inversions. The biased endpoint selection may reflect the limited search for target DNA sequences by the Tn10 transposase, and the spatial proximity of the donor and target regions in the folded S. typhimurium nucleoid. Using this interpretation, the data suggest that DNA sequences 40-80 kb clockwise and counterclockwise of the insertion site are in spatial proximity with the insertion, perhaps reflecting the organization of DNA into ~120-kb nucleoid domains. In addition, the data predict the spatial proximity of several distant DNA regions, including DNA sequences equidistant from the origin of DNA replication.     

Two complementation groups mediate tetracycline resistance determined by Tn10.

The structural and regulatory tetracycline resistance genes of transposon Tn10 are located on a 2,700-base pair HpaI fragment. We have used eight tetracycline-sensitive mutations in the 2,700-base pair fragment, cloned into two compatible plasmids, to demonstrate that two complementation groups are required for tetracycline resistance. By genetic recombination with plasmids containing the regulatory or structural regions for resistance, we have determined that both complementation groups reside within the structural region. The complementation groups, designated tetA and tetB, are proximal and distal, respectively, to the promoter for the tetracycline resistance structural region. The tetB mutations are in the portion of the structural region that is known to encode the 36,000-molecular-weight, inner-membrane TET protein. The levels of tetracycline resistance expressed during complementation suggest a complex interaction between the products of the tetA and tetB loci.     

Tn10 generated deletions of the ompB locus of Escherichia coli K12

Abstract We have isolated a set of Tn 10 -generated deletions starting from the distal end of the ompR envZ operon of Escherichia coli K12. Most of the deletions removed both ompR and envZ genes or ended in ompR . These deletions exhibited an OmpC⁻ OmpF⁻ phenotype. One deletion removed only part of envZ and the strain was phenotypically OmpC⁻ OmpF^+/−. This deletion of the distal part of envZ did not affect osmoregulation of ompC . However, ompF osmoregulation appeared reversed. High osmolarity in the growth medium resulted in production of OmpF close to the wild-type level.     

Intracistronic complementation of the tetracycline resistance membrane protein of Tn10.

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.     

Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element

Summary Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield trp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of trp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of -transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final ::Tn409 or ::Tn410 product.Abbreviations p.f.u. plaque-forming units - MIC minimal inhibitory concentration - LFT low frequency transducing - HFT high frequency transducing     

Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control

Binding of the Tet repressor to nonspecific and specific DNA leads to quenching of the Tet fluorescence by approximately 22% and approximately 35%, respectively. This effect is used for a direct, quantitative characterization of the binding equilibria and dynamics involved in the recognition of the operator by its repressor. From the dependence of the nonspecific binding constant on the ion concentration, it is concluded that nonspecific binding is almost completely driven by the entropy change resulting from the release of three to four Na+ ions from the double helix upon protein binding. Formation of the specific complex is driven by a higher entropy term resulting from the release of seven to eight Na+ ions and in addition by a free energy term of -33 kJ/mol from nonelectrostatic interactions, which are attributed to the specific contacts. The dynamics of the repressor-operator recognition are resolved by stopped-flow measurements at various salt concentrations and for different DNA chain lengths into two separate steps. The first step follows a second-order mechanism and results in an intermediate complex associated with formation of about three to four electrostatic contacts between protein and DNA; apparently, this complex is equivalent to the nonspecific complex. The existence of an intermediate is also indicated by experiments in mixed Na+-Mg2+ buffers, which can be described with high accuracy by competition of Mg2+ and protein. The intermediate complex is formed at a rate of 3 X 10(8) M-1 s-1 and is converted in the second reaction step to the specific complex with a rate constant of 6 X 10(4) s-1, which is almost independent of the salt concentration. Our interpretation and the parameters obtained from our model are confirmed by competition of nonspecific DNA with operator DNA for repressor binding. The observed maximal rate constant of 3 X 10(8) M-1 s-1 is very close to theoretical predictions for the association without a sliding mechanism. The very small dependence of the observed rate constants on the chain length shows that the Tet repressor is not able to slide over any substantial distance even at low salt concentrations. The question of a potential contribution from sliding under our experimental conditions is critically discussed. The absence of sliding in the case of the Tet repressor under physiological conditions is compared with the high sliding efficiency of the lac repressor and is discussed with respect to possible molecular mechanisms of sliding in relation to biological function.     

Location of ribosome-binding sites on the tetracycline resistance transposon Tn10.

Eight ribosome-binding sites were located on the single-stranded Tn10 DNA loop which was formed after denaturation of lambda phage DNA containing the Tn10 transposon sequence. Ribosomes were bound only to the Tn10 loop contained on the R strand of lambda DNA but not to that on the L strand, suggesting that one of the two strands of Tn10 DNA is selectively transcribed. Six of the eight ribosome binding sites were located in one-half of the DNA loop. The maximum sizes of potential polypeptides were calculated for these genes to range between 9,500 and 84,000 daltons.     

Identification of the tetracycline resistance promoter and repressor in transposon Tn10.

The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.     

Nearly precise excision: a new type of DNA alteration associated with the translocatable element Tn10.

We describe an unusual DNA alteration, "nearly precise excision," which has been identified among tetracycline-sensitive deletion derivatives of lambda phages carrying the translocatable tetracycline-resistance element Tn10. DNA sequence analysis of two such derivatives demonstrates that each retains exactly 50 bp of Tn10 material. The original junctions between lambda and Tn10 sequences remain intact; however, an internal deletion has occurred within Tn10 which eliminates all but the last few base pairs at each end of the element. This deletion occurs within a short A + T-rich inverted repeat which is present near each end of Tn10. Nearly precise excisions occur at frequencies comparable to Tn10-promoted deletions, inversions and translocations, and, like these other events, are independent of phage and bacterial functions for homologous recombination (recA, recB, red). It is not yet clear, however, whether nearly precise excisions are specifically promoted by Tn10 or whether they arise during the course of normal DNA replication processes as a consequence of unusual symmetries present in the DNA sequence at the ends of Tn10.     

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.     

Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10

The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Deletions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry.     

Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae.

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.     

recA-dependent genetic switch generated by transposon Tn10

We describe a new type of regulatory switch generated in bacteriophage lambda by transposon Tn10. By this switch, phage genes alternate reversibly between expressed and non-expressed states as the direct consequence of a reversible DNA rearrangement. The switch itself has arisen via Tn10-promoted recombination. The subsequent “flip-flop” in gene expression occurs by general recombination between two IS10 elements serving as “portable regions of homology”.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

Characterization of Tn916S, a Tn916-like element containing the tetracycline resistance determinant tet(S)

We have characterized a transferable tetracycline resistance (Tcr) element from a Streptococcus intermedius isolate. The gene responsible for this resistance was identified by PCR and Southern hybridization as tet(S). Furthermore, the genetic support for this determinant was shown to be a conjugative transposon closely related to Tn916. This element has been designated Tn916S.     

Constitutive expression of tetracycline resistance mediated by a Tn10-like element in Haemophilus parainfluenzae results from a mutation in the repressor gene.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.     

Physical mapping of the srl recA region of Escherichia coli: analysis of Tn10 generated insertions and deletions

Summary A restriction endonuclease map for the enzymes EcoRI, BamHI, SalI, and PstI covering 23.5 kilobase pairs (kb) of the srl recA region of Escherichia coli was constructed. An insertion of the transposon Tn10 in the negative regulatory gene srlR was shown to be located 5.8 kb away from the promoter roximal end of the recA gene. The extent of several Tn10 generated deletions, originating from the srlR301::Tn10 insertion, were analyzed by physical mapping. Three mutations that had removed the Tn10 encoded tetracycline resistance gene, del(srl-recA)302, del(srl-recA)304, and del(srl-recA)303, were found to be deleted for 40%, 45% and 50% of the recA structural gene, respectively. A deletion, del(srl-recA)306, that had not affected the structure of the Tn10 in srlR301 was shown to have removed the entire recA structural gene.