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.     

Sucrose utilization determinants of transposon Tn2555

The Sac- -derivatives of the plasmid pBRS5.2 containing the sucrose utilization transposon Tn2555 were obtained by using the insertional mutagenesis. The sac mutations were mapped within the transposons DNA fragment of about 3kb. The existence of two linkage groups of the sac mutations was shown by complementation analysis. Among the gene products of Tn2555 the polypeptides of about 58, 36, 27, 14 and 12 kd were found. The data on relation of the sac genes of Tn2555 and the ones of the known plasmid pUR400 of Salmonella typhimurium are discussed.     

Structure and mode of transposition of Tn2555 carrying sucrose utilization genes

The sucrose transposon Tn2555 from Escherichia coli, which has an unstable structure, was studied in more detail. Sequence analysis of one of the transposon variants, designated Tn2555.3, revealed the presence of two direct IS26 copies on its flanks, and a third inverted IS26 copy inside the transposon. The sucrose utilization genes of Tn2555.3 were found to be identical to those of the previously described pUR400 plasmid. It was demonstrated that Tn2555.3 translocation from pBR325 to RP4 occurs via a cointegrate formation, mediated by one of the three IS26 copies, followed by its resolution due to RecA-dependent recombination between two direct IS26 copies flanking the donor replicon.     

Genetic organization of transposon Tn10

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.     

Structural and functional analyses of the fosfomycin resistance transposon Tn2921.

The fosfomycin resistance transposon Tn2921 is flanked by directly repeated sequences homologous to the Tn10-related insertion sequence IS10. The nonrepeated DNA sequences of Tn2921 can be deleted without affecting the transposition ability of the element, showing that at least one of the direct repeats is an active insertion sequence. Transposition of Tn2921 seems to occur through direct transposition, since cointegrates have not been observed. The evolutionary relatedness of Tn2921 and IS10 is discussed.     

Genetic organization of the bacterial conjugative transposon Tn916.

Tn916, which encodes resistance to tetracycline, is a 16.4-kilobase conjugative transposon originally identified on the chromosome of Streptococcus faecalis DS16. The transposon has been cloned in Escherichia coli on plasmid vectors, where it expresses tetracycline resistance; it can be reintroduced into S. faecalis via protoplast transformation. We have used a lambda::Tn5 bacteriophage delivery system to introduce Tn5 into numerous sites within Tn916. The Tn5 insertions had various effects on the behavior of Tn916. Some insertions eliminated conjugative transposition but not intracellular transposition, and others eliminated an excision step believed to be essential for both types of transposition. A few inserts had no effect on transposon behavior. Functions were mapped to specific regions on the transposon.     

Tn951: A new transposon carrying a lactose operon

Summary A new transposon, Tn951, is described, which derives from plasmid pGC1, originally isolated from Yersinia enterocolitica. Tn951 is 16.6 kb long and presumably flanked by small inverted repeats. It carries the lac genes i, z and y. This lac system is homologous to the E. coli lac operon. However, homology is restricted to 5.6 kb. The DNA sequences surrounding the lac operons on Tn951 and E. coli are nonhomologous. This leads to speculations about the origin of the E. coli lac operon itself.     

The organization of the outside end of transposon Tn5.

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.     

Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.     

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.     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.     

Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain.     

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.     

Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens

The effects of kanamycin and streptomycin added to soil on the survival of transposon Tn5 modified Pseudomonas fluorescens strain R2f were investigated. Kanamycin in high (180 g g^-1 dry soil) or low (18 g g^-1) concentration or streptomycin in low concentration in Ede loamy sand soil had no noticeable effect on inoculant population dynamics in soil and wheat rhizosphere, whereas streptomycin in high concentration had a consistent significant stimulatory effect, in particular in the wheat rhizosphere. Streptomycin exerted its effect by selecting P. fluorescens with Tn5 insertion whilst suppressing the unmodified sensitive parent strain, as evidenced by comparing the behaviour of these two strains in separate and mixed inoculation studies.Soil textural type influenced the effect of streptomycin on the Tn5 carrying inoculant; the effect was consistently detected in rhizosphere and rhizoplane samples of wheat grown in Ede loamy sand after 7 and 14 days incubation, whereas it was only apparent after 7 days in rhizoplane or rhizosphere (and bulk soil) samples of wheat grown in two silt loam soils. Modification of soil pH by the addition of CaCO₃ or bentonite clay resulted in an enhancement of the selective effect of streptomycin by CaCO₃ and its abolishment by bentonite clay.The addition to soil of malic acid or wheat root exudate, but not of glucose, enhanced the streptomycin selective effect on the Tn5-modified P. fluorescens strain. Neither the streptomycin producer Streptomyces griseus nor two non-inhibiting mutants obtained following UV irradiation affected the dynamics of P. fluorescens (chr::Tn5) in soil and wheat rhizosphere.The effect of streptomycin in soil on inoculant Tn5 carrying bacteria depends on conditions such as soil type, the presence of (wheat) root exudates and the type of available substrate.     

Identification of additional genes on transposon Tn10: tetC and tetD.

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).     

Characterization of transposon Tn5086, carrying the site-specifically inserted gene dhfrVII mediating trimethoprim resistance.

Two different enteric plasmids of widely separate origins were observed to carry a new 15.3-kb trimethoprim resistance transposon, Tn5086, also mediating resistance to mercuric ions and to a low level of sulfonamide. The trimethoprim resistance gene characterized from Tn5086 was found to be distinct from those found earlier and was designated type VII. Molecular analysis demonstrated that Tn5086 is closely related to Tn21. The internal part of Tn21 and Tn5086, the element referred to as the integron, was found to be different. First, the integron of Tn5086 contains a 0.62-kb cassette formed by the trimethoprim resistance gene dhfrVII and its immediate surroundings instead of the 0.86-kb aadA1 cassette of Tn21. Second, the integron of Tn5086 lacks a 4.2-kb segment 3' of sulI in Tn21. The dhfrVII gene commences with a UUG codon but was otherwise seen to be markedly related to the cassette genes dhfrI, dhfrV, and dhfrVI. The four related dihydrofolate reductases of 157 amino acids encoded by these genes contain a glutamate instead of the aspartic acid residue found at position 27 of the active center of the chromosomal enzyme from Escherichia coli.     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.