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.     

Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.     

Multiple integration sites for the lactose transposon Tn951 on plasmid RP1 and establishment of a coordinate system for Tn951

Summary Various molecules generated by transposition of the lactose transposon Tn951 from plasmid pGC1 to plasmid RP1 were examined by DNA heteroduplex and restriction endonuclease analysis. Tn951 was found to transpose to at least eight different sites on RP1 in both possible orientations. A coordinate system for the lactose transposon Tn951 is constructed.     

Expression and regulation of the lactose transposon Tn951 in Rhizobium spp.

Abstract The expression of β-galactosidase by the lac transposon Tn 951 , in Escherichia coli , was found to be cAMP-dependent. This finding provided the basis for an investigation of the effect of cAMP on Tn 951 lac expression in Rhizobium , with the ultimate aim of using the Tn 951 system as a specific probe for cAMP mediated catabolite repression. When introduced into Rhizobium , Tn 951 directed the synthesis of β-galactosidase, which was inducible by isopropyl-β- d -thiogalactopyranoside (IPTG). Marked quantitative and qualitative differences in β-galactosidase expression were found between R. meliloti and R. japonicum during the growth cycle, with expression being higher in the former. β-Galactosidase levels were, however, unaffected by exogenous cAMP under catabolite repressing conditions.     

Deletions and an inversion induced by a resident IS1 of the lactose transposon Tn951

Summary DNA-DNA filter binding tests, Southern blotting experiments and DNA heteroduplex analysis clearly show that Tn951 contains an IS1 element. This IS1-951 sequence is peculiar in that it does not contain the PstI cleavage site which is usually observed on E. coli derived IS1 elements. Nonetheless, IS1-951 induces deletions. This process is temperature dependent. One instance of an IS1-951 induced inversion was observed, the structure of which is compatible with the current models of transposition of IS elements.     

Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919

Tn919 is a 15- to 16-kilobase (kb) tetracycline resistance conjugative transposon that was originally isolated from Streptococcus sanguis FC1. The tetracycline resistance determinant (tet) was found on a 4.2-kb HindII fragment by in vitro deletion analysis. This fragment was subcloned to a pWV01 origin capable of directing replication in Escherichia coli, Bacillus subtilis, and Streptococcus lactis, and expression was observed in all three genera. In all cases, expression was weaker when only the 4.2-kb cloned fragment rather than the full transposon was present. The resistance gene is of the streptococcal tetM class and codes for a protein of approximately 70 kilodaltons. The restriction map resembles that of the tetM gene of Tn1545 (P. Martin, P. Trieu-Cuot, and P. Courvalin, Nucleic Acids Res. 14:7047-7058, 1986), which codes for a protein of 72.5 kilodaltons. A number of transposon-derived promoter-bearing fragments were also cloned and sequenced. These closely resemble the consensus sequence of E. coli and B. subtilis promoters. Fusion experiments with a truncated lacZ gene indicate the possibility of an open reading frame for one of the promoters.     

Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1.

Tge transposon Tn21 has been transposed from R100.1 to plasmid pACYC184 and, from the resulting recombinants, to plasmid R388. The sites of insertion and the orientation of the element in several pACYC184::Tn21 recombinants have been examined. Restriction enzyme analysis of these recombinants has resulted in a detailed map of Tn21; this is compared with the published maps of the relevant part of R100.1. Heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. With various in vitro-generated deletion mutants of Tn21, the internal gene necessary for transposition (tnpA) was localized within the terminal 4.3 kilobases of the right-hand end of the element. Genetic analysis of transposition of Tn21 suggests that the process proceeds via cointegrates. Since the end products of transposition are simple recombinants of the element and the recipient replicon, Tn21 must contain a gene that codes for a resolvase type of activity (tnpR gene).     

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.     

Tn5037, a Tn21-like Mercury Resistance transposon from Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidanswas cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21branch of the Tn21subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037is organized similarly to most of the Gram-negative bacteria meroperons and is closest to that of ThiobacillusT3.2. The operator-promoter region of the meroperon of Tn5037also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidansE-15, and ThiobacillusT3.2, respectively. No inducibility of the Tn5037 meroperon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037was inactive in Escherichia coliK12, in contrast to its resolution system (ressite plus gene tnpR). However, transposition of Tn5037in this host was provided by the tnpAgene of Tn5036, a member of the Tn21subgroup. Sequence analysis of the Tn5037 ressite suggested its recombinant nature.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

A Tn21 terminal sequence within Tn501: complementation of tnpA gene function and transposon evolution

Summary The prokaryotic mercury-resistance transposon Tn501 contains a sequence, 80 nucleotides from one end, which is identical with an inverted terminal repeat (IR) of Tn21. This Tn21 IR sequence is used when Tn21 complements a TnpA^- derivative of Tn501, but not when Tn501 is used for the complementation. Complementation by Tn1721 shows a preference for the normal Tn501 IRs. The element (Tn820) transposed when Tn21 is used to complement a Hg^- TnpR^- TnpA^- Res^- deletion mutant of Tn501 contains the Tn21 IR sequence at one terminus and a Tn501 IR at the other. Transposition of Tn820 can be complemented by Tn501 and Tn1721, but at a much lower frequency than transposition of the parental element (Tn819) which has two Tn501 IRs. The relationship between the transposition functions of Tn501, Tn21 and Tn1721, and available nucleotide sequence data suggest that Tn501 evolved by the transposition of a Tn21-like element into another transposable element (similar to that found within Tn1721) followed by deletion of the Tn21-like transposition functions.Abbreviations used (IR) Inverted repeat - (Cb) carbenicillin - (Cm) chloramphenicol - (Sm) streptomycin - (Su) sulphonamide - (Tc) tetracycline - (Tp) trimethoprim     

Construction and characterization of versatile kanamycin-resistance cassettes derived from the Tn5 transposon

We have developed plasmids with the Tn5 kanamycin-resistance gene (kan) flanked either symmetrically or asymmetrically by several restriction sites. These can be used to provide a selectable genetic marker or to mobilize restriction sites and sense or nonsense codons into genes. The 1.3-kb kan cassette exhibits polarity effects in both directions.     

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.     

Requirements for strand- and site-specific cleavage within the oriT region of Tn4399, a mobilizing transposon from Bacteroides fragilis.

Replicons that contain Tn4399, a conjugal mobilizing transposon isolated from Bacteroides fragilis, can be mobilized in the presence of broad-host-range IncP plasmids RP4 and R751 in Escherichia coli to B. fragilis or E. coli recipients (C. G. Murphy and M. H. Malamy, J. Bacteriol. 175:5814-5823, 1993). To identify the initial DNA processing events involved in Tn4399-mediated mobilization in E. coli, plasmid DNA from pCGM328 (a pUC7 vector that contains the mobilization region of Tn4399) was isolated from donor cells following the release of plasmid DNA from the relaxation complex. Site- and strand-specific cleavage within the oriT region of Tn4399 was detected by denaturing gel electrophoresis and Southern hybridization analysis of this DNA in the presence or absence of IncP plasmids. Mutations in either mocA or mocB, two genes which are encoded by Tn4399 and are required for mobilization, significantly decrease the amount of specifically nicked DNA detected. These results suggest roles for the MocA and MocB gene products in specific processing of Tn4399-containing plasmid DNA prior to mobilization. By isolation of the nicked strand and primer extension of this template, we mapped the precise 5' end of the single-stranded cleavage reaction. The nucleotide position of nicTn4399 is adjacent to two sets of inverted repeats, a genetic arrangement similar to those of previously characterized oriT regions. Two site-directed mutations which remove nicTn4399 (oriT delta 1 and oriT delta 2) cannot be mobilized to recipients when they are present in trans along with functional MocA and MocB proteins and an IncP mobilizing plasmid; they are cis-dominant loss-of-function mutations.     

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.     

Identification of Tn2401, a transposon encoding multiresistance to aminoglycosides

A transposable element, Tn2401, was found in a clinical isolate of Pseudomonas aeruginosa. Tn2401 had a size of 7190 nucleotides and encoded aminoglycoside 3'-phosphotransferase and aminoglycoside 6'-N-acetyltransferase. The sequence encoding the former enzyme was homologous with that of Tn903. Pseudomonas aeruginosa strains harbouring this transposon were resistant to kanamycin, neomycin, lividomycin, ribostamycin, paromomycin, netilmycin, tobramycin, dibekacin, gentamicin, sisomicin, and butirosin.     

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.

Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400 delta BglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400 delta BglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400 delta BglII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.