Transposon Tn554 encodes three products required for transposition.

Tn554 is a high-frequency, site-specific, transposable element having integrative properties resembling those of lysogenic bacteriophages. Nucleotide sequence analysis indicates that Tn554 has three transposition genes, designated tnpA, tnpB and tnpC. Mutations in each of these were complemented efficiently in trans by clones containing internal fragments of Tn554; thus the products of these genes function in trans. Elements carrying deletions of the Tn554 termini could not be complemented. The product of tnpC is not absolutely required for transposition, since deletion mutations encompassing 80% of tnpC, as well as frameshift mutations located near the amino terminus of tnpC, transposed at frequencies as high as 2% of that observed with wild-type Tn554. However, such mutations affected the orientation of insertion. With wild-type Tn554 insertion occurs in a single orientation regardless of the orientation of the donor. In tnpC mutants insertion orientation was dictated by the orientation of Tn554 in the donor molecule. A mutant lacking the carboxy-terminal 59 residues of tnpB also exhibited altered insertion orientation. Thus it appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy-terminal portion of the tnpB gene product.     

Transposon Tn4556 of Streptomyces fradiae: nucleotide sequence of the ends and the target sites.

A transposon, Tn4556, has recently been isolated from Streptomyces fradiae (S.-T. Chung, J. Bacteriol. 169:4436-4441, 1987). The ends of Tn4556 were found to contain inverted repeats of 38 base pairs with 70% sequence identity with the ends of Tn3. Insertion of Tn4556 into a Streptomyces plasmid resulted in a 5-base-pair duplication of the target site.     

Transposon Tn5403, a mobilization-helper element: Complete nucleotide sequence and distribution in aquatic strains

Abstract Tn 5403 , a new transposable element, was found in a Klebsiella pneumoniae strain (KIIIA) isolated from an aquatic environment following a pollution accident. It was identified by its ability to mobilize ori T-deleted recombinant plasmids and thus can be proposed as a ‘helper’ element participating in the transfer mechanisms of non-conjugative plasmids. Nucleotides sequence analysis shows that the element consists of 3663 bp. Two open reading frames (ORFs) are found: one ( tnp A) encoding a putative transposase and the other ( tnp R) a putative resolvase. The two ORFs are transcribed in opposite directions and are separated by a putative recombination site (res). Results obtained with a specific probe suggest that Tn 5403 or a similar element is present in different Enterobacteriaceae strains isolated from the same river three years after the isolation of the Tn 5403 -harbouring KIIIA strain.     

Analysis of the complete nucleotide sequence of the tetracycline-resistance transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10(R100), demonstrating the conservation that exists between these transposons. A previously uncharacterized approximately 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

Complete nucleotide sequence of Tn10

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.     

Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance

Summary We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.     

Tn554: Isolation and characterization of plasmid insertions

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition of transposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, φ11, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”     

The nucleotide sequence of the tnpA gene of Tn21.

The nucleotide sequence of the tnpA gene of Tn21 is presented. The transposase encoded by this gene is exactly the same length (988 amino acids) as the Tn501 transposase (4), and shows 72% homology overall with this protein, with greater homology towards the C-terminus. The sequence of the transposase is discussed in the context of the evolution of Class II transposable elements and of the characteristics of the enzyme's action.     

Transposon mutagenesis in a marine synechococcus strain: isolation of swimming motility mutants

Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique type of swimming motility characterized by the absence of flagella or any other obvious organelles of motility. While the abundant cell surface-associated 130-kDa glycoprotein SwmA is known to be required for the generation of thrust, identification of other components of the motility apparatus has, until recently, been unsuccessful. Here we report on the development of a transposon mutagenesis system for use with marine Synechococcus sp. strain WH8102, a model organism for which the genome has been sequenced. Utilizing this mutagenesis technique, we have isolated 17 independent mutants impaired in swimming motility. These 17 transposon insertions are located in nine open reading frames, which cluster in three separate regions of the genome. Included within these clusters are several multicomponent transport systems as well as a number of glycosyltransferases.     

Transposon Tn916 mutagenesis in Clostridium botulinum.

The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10(-8) to 10(-5) Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindIII chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.     

Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants

When the "suicide" vector pSUP1011, which carries transposon Tn5 (Kmr), was introduced into Rhizobium japonicum USDA 110, kanamycin-resistant (Kmr) colonies were detected at a frequency (4.2 X 10-6) ca. 30 times greater than the spontaneous kanamycin resistance frequency (1.4 X 10-7). Ten thousand Kmr mutants were isolated and tested for nutritional auxotrophy. Auxotrophs were detected at a frequency of 0.5%. The following classes of auxotrophs were identified: adenine- (three), histidine- (three), glutamate- (five), adenine plus thiamine- (nine), uracil- (three), pantothenic acid- (one), tryptophan- (three), and methionine- (three). Mutants blocked in symbiotic nitrogen fixation (Fix-) were also identified at a frequency of 3%. The glutamate auxotrophs were studied in more detail, and all five showed an altered expression of nitrogenase activity in free-living cultures.     

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

Mutational analysis of att554, the target of the site-specific transposon Tn554.

Tn554 is a high-frequency, site-specific transposable element of Staphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, 5'-GATGTA-3' (nucleotides numbered -3 to +3). Most of the time (greater than 99%) insertion occurs immediately 3' to this sequence; the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5' to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the left and ending at -15 or from the right ending between +8 and +12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active att554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequences at the junction of att554 and the 3' end of Tn554. These data indicate that att554 contains a short nucleotide sequence essential for transposition and flanking sequences that greatly increase the frequency of recombination.     

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.