Transposition of TnA does not generate deletions

Summary We have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon, R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.     

Mapping of functions in the R-plasmid R388 by examination of deletion mutants generated in vitro.

Mutant plasmids in which large segments of R388 DNA are deleted were constructed in vitro from two R388::TnA (Tn801) plasmids, using the BamHI site of TnA and the BamHI and BglII sites of R388. These deletion mutants permitted mapping of genetic functions into the restriction map of R388.     

Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K.

Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA.     

Properties of derivatives of the Pseudomonas plasmid pVS1 that have inherited carbenicillin resistance from RP1.

A procedure is described for the isolation, in Pseudomonas aeruginosa PAO, of bacteria carrying derivatives of pVS1 that inherited the carbenicillin-resistance determinant from RP1 either alone or together with that for aeruginocin resistance. Such bacteria occur among the transconjugant progeny from both recombination-proficient or -deficient pVS1+ RP1+ donors, suggesting that the formation of these plasmids is due to the translocation of TnA from RP1 into pVS1. It is possible, therefore, that the aeruginocin-resistance determinant is part of TnA or is closely linked to it. Unexpectedly, none of these plasmids showed the 3 x 10(6)- to 4 x 10(6)-dalton increase in size predicted for TnA+ derivatives of PVS1. It is suggested that an interaction between TnA and the Tn501 translocation unit in pVS1 could account for this result.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

Modulation of mutagenesis involving precise excision of transposon Tn10

Precise excision of transposon Tn10 results in reversion of the Trp- phenotype to Trp+ in a trp-1014::Tn10 strain of Salmonella typhimurium, and also occurs at a markedly higher frequency in a strain carrying the temperature-sensitive polA7 allele. The frequency with which precise excision events occurs can be modified by the plating medium, results indicating that the great majority of mutants which arise on broth-supplemented or tryptophan-supplemented minimal media actually arise on the selective plating medium. Trp+ revertants (1000) arising from excision of Tn10 were purified by re-streaking for single colonies; none were found to retain the Tn10 encoded resistance to tetracycline. Yields of Trp+ revertants of the polA7 strain were consistently higher when glycerol rather than glucose was used as sole carbon source in the selective medium. Clean excision of Tn10 can also be increased by ultraviolet irradiation in (R) plasmid-free strains, and is further increased in strains carrying an N-group plasmid (R205, R46 or pKM101). Ultraviolet-induced precise excision of Tn10 also occurs at a much enhanced frequency in a strain with a deletion through the uvrB gene; in this case, however, the addition of plasmid pKM101 leads to a decrease in yields of ultraviolet-induced precise excision events.     

Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1.

The region of plasmid NR1 concerned with resistance to Hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColE1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColE1, cells containing the plasmid was as sensitive to Hg2+ and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColE1, cells with the hybrid plasmid were hypersensitive to Hg2+ and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).     

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.     

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Tn1 generated mutants in the mercuric ion reductase of the Inc P plasmid, R702

Summary Physiological, biochemical and genetic aspects of resistance to inorganic mercury compounds were examined in a group of mercury sensitive derivatives generated in the Inc P plasmid, R702, by Tn1 insertion. Strains carrying each of these insertion mutations had no detectable mercuric ion reductase, were more sensitive to mercuric ion than a plasmidless strain, and exhibited inducible uptake of Hg²⁺. These characteristics indicate that the mutants are altered in the Hg(II) reductase. This hypothesis was supported by complementation and recombination analysis with known point and deletion mutations in the mer operon of the Inc FII plasmid, R100. Such experiments showed that the eight insertions studied had occurred in four distinct regions of the Hg(II) reductase structural gene (merA). Complementation data also demonstrated that the regulatory protein determined by the R702 plasmid has no effect on the expression of the micro-constitutive Hg(II) reductase activity expressed by merR mutants of R100.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Regional preference of insertion of Tn501 and Tn802 into RP1 and its derivatives

Summary The sites of insertion of Tn501 into RP1 and into derivatives of this plasmid that either lack the Tn801 (TnA) element or contain it in a different location have been determined. Similarly, the sites of insertion of Tn802 into a derivative of RP1 that lacks the Tn801 element and into recombinants of this plasmid with Tn501 were determined. Hot spots for insertion were observed with both transposons; but it is clear that a particular DNA sequence is not sufficient to define a hot spot, since a particular region does contain many insertions when present in one plasmid but does not do so when part of another.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b

Tn7 insertion mutagenesis has been used to facilitate the generation of a physical (restriction endonuclease) and genetic map of the IncM plasmid, R831b. The only selectable phenotypes carried by this 90-kb conjugative plasmid are resistances to inorganic mercury [Hg(II)] and to organomercury compounds. Mutants in the Hgr locus of R831b complemented previously described mutants in the mer operon of the IncFII plasmid R100, indicating functional homology of the locus in each of these different plasmids. However, the R831b Hgr locus is not notably similar in restriction site pattern to either the mer operon of R100 or the mercury resistance transposon, Tn501. Although the enzymes they encode are co-ordinately regulated, the Omr locus of R831b maps approx. 13.5 kb away from the Hgr locus. Three insertions which affect neither phenotype lie between the Hgr and Omr loci; thus, the loci are separated both physically and genetically. One mutant was obtained which tentatively identifies the position of the Tra locus of R831b as adjacent to the Hgr locus.     

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.     

Sensitivity to antimicrobial agents of some mercury-sensitive and mercury-resistant strains of Gram-negative bacteria

The sensitivity and resistance of some Gram-negative mercury (Hg²⁺)-sensitive and-resistant strains to chemotherapeutic agents and to disinfectants and preservatives are described.Escherichia coli andPseudomonas aeruginosa strains harboring plasmid pUB 1351 [pUB 367:Tn 501] andE. coli bearing R100-1 were resistant to inorganic mercury and to various antibiotics, but were not more resistant to organic mercury and other preservatives and disinfectants than plasmidless strains.