Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Genetic characteristics of plasmid pAP53 derepressed in transfer functions detected in the cells of an opportunistic E. coli strain

A study was made of plasmid pAP53 derepressed as regards transfer functions (Tra-functions) detected in E. coli strain cells, serogroup 0128, after its labeling with transpozones Tn1 and Tn9. The compatibility tests demonstrated that the plasmid belongs to the incompatibility group FIII and is partially incompatible with the group FII reference-plasmid. Plasmid pAP53 is unable to inhibit Tra-functions of plasmid F'lac and is not inhibited by the fin type genetic regulation on the OP group plasmids under study. At the same time Tra-functions of plasmid pAP53 are inhibited in the presence of pAP41 plasmid, which indicates that this plasmid has a special type of genetic regulation.     

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.     

Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element.

A procedure is described that selects for the insertion of transposable antibiotic resistance elements in a variety of recipient replicons. The selected translocation procedure, which employs a plasmid having a temperature-sensitive defect in replication as a donor of transposable genetic elements, was used to investigate certain characteristics of the translocation process. Our results indicate that translocation of the Tn3 element from plasmid to plasmid occurs at a 10(3)- to 10(4)-times-higher frequency than from plasmid to chromosome. In both cases, continued accumulation of Tn3 on recipient genomes is prevented by development of an apparent equilibrium when only a small fraction of molecules in the recipient population contain Tn3. An alternative method for estimation of translocation frequency has shown that the translocation process is temperature sensitive and that its frequency is unaffected by the presence of host recA mutation. Insertions of Tn3 onto the 65 X 10(6)-dalton R6-5 plasmid in Escherichia coli are clustered on EcoRI fragments 3 (8 of 23 insertions) and 9 (7 of 23 insertions), which contain 12 and 5%, respectively, of the R6-5 genome. The occurrence of multiple insertions of Tn3 within EcoRI fragment 9, which contains the IS1 element and a terminus of the Tn4 element, is consistent with earlier evidence indicating that terminal deoxyribonucleic acid sequences of already present transposable elements may provide recognition sequences for subsequent illegitimate recombinational events.     

Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245

The expressed difference in the plasmid profile of A. brasilense Sp245 is registered as a result of Tn5-Mob-mutability. Integration of the vector pSUP5011 into one of the A. brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported. The properties of A. brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed. The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium. The results suppose 85Md plasmid participation in melaninogenesis.     

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.     

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.     

Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Transposition of Tn7 in Pseudomonas aeruginosa and Isolation of alk::Tn7 Mutations

Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.     

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.     

Characterisation of Tn1721, a new transposon containing tetracycline resistance genes capable of amplification.

R plasmid pRSD1 contains tetracycline resistance (tet) genes in a 3.55 Mdal-region capable of amplification by forming tandem repeats (Mattes, Burkardt and Schmitt, Molec. gen. Genet., 1979). The repetitious tet element is itself part of a 7.2 Mdal-transposon, named Tn1721, as demonstrated by the following criteria; (i) Tn1721 has been translocated to phage lambda. The resulting hybrid phage lambda tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not continguous with it indicating translocation of the element by non-homologous recombination. In addition, lambda tet has sustained a 3.4 Mdal-deletion adjacent to the insertion. (ii) Further transposition of Tn1721 to the 21.5 Mdal-plasmid R388 resulted in R388::Tn1721 derivatives, two of which were characterised. They contain Tn1721 inserted into different sites but in the same orientation as shown by restriction and heteroduplex analyses. These translocation of Tn1721 were not accompanied by deletions of DNA. (iii) The insertion plasmid pRSD102(R388::Tn1721) has conserved the capacity of the original plasmid pRSD1 to amplify the 3.55 Mdal-tet region. It has been concluded that Tn1721 constitutes a novel transposon encompassing a tet region capable of selective amplification. The model proposed for Tn1721 contains three short repeats. Two direct repeats, flanking the 3.55 Mdal tet region, provide sequence homology for amplification. The third repeat (located distally to tet) is inverted and provides the basis for transposition of the 7.2 Mdal-element.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem.

A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.     

pλCM System: Observations on the Roles of Transposable Elements in Formation and Breakdown of Plasmids Derived from Bacteriophage Lambda Replicons

Transduction with phage derived from a 2-year-old lysate of lambda cam105 (lambda::Tn9) gave rise to chloramphenicol-resistant (Cm(r)) transductants harboring a plasmid (plambdaCM1) formed from lambda cam105 by a Tn9-mediated adjacent deletion to position 36.07 kilobases in the N cistron of lambda. The plambdaCM element can replicate as a plasmid, insert into the bacterial genome, or reproduce lytically as a phage on cells that provide N function. The feasibility of obtaining high titers in encapsidated form and the ease of synchronous introduction into and recovery from bacterial populations make plambdaCM very suitable for quantitative studies of recombination involving transposable elements. Replicon fusions between plambdaCM1 and RSF1596 (pMB8::Tn3Delta596) occur by duplication of either IS1 (at low rate in the absence of TnpA activity) or Tn3Delta596 (in the presence of TnpA activity). At 24 or 32 degrees C, the rate of increase of TnpA-mediated fusions per plambdaCM is about 2% per cell doubling. RSF103 contains the deleted Tn1DeltaAp (which lacks intact beta-lactamase and TnpR resolvase coding sequences) adjacent to a streptomycin resistance (Sm(r)) determinant. We observed that Tn1DeltaAp mediates insertions of external RSF103 sequences into the R388 plasmid. R388::Tn1DeltaAp plasmids show transposition immunity in cells lacking TnpR activity. Using the plambdaCM system, we isolated adjacent transpositions of the RSF103 Sm(r) determinant. The resulting plambdaCM-Sm cosmids contain Sm(r) genetic material flanked by direct repeats of Tn1DeltaAp, and all are deleted for some RSF103 or plambdaCM sequences. The plambdaCM-Sm constructs will fuse into R388 by duplication of a single Tn1DeltaAp element. In the presence of tnpR(+) (but not tnpR) Tn1 or Tn3 elements, all Tn1DeltaAp-mediated complex replicons break down completely and rapidly to simple Tn1DeltaAp inserts. The equilibrium for resolution is at least 10(5):1, and resolution is more than 90% complete after 40 min of exposure to a tnpR(+) cytoplasm. In the absence of TnpR, Rec, and Red activities, Tn1DeltaAp-mediated complex replicons yield simple Tn1DeltaAp inserts at a lower rate. The presence of intact RSF103 replication determinants between direct Tn1DeltaAp repeats appears to accelerate this precise TnpR- and Rec-independent breakdown.     

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.