Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

Transconjugant analysis: limitations on the use of sequence-specific endonucleases for plasmid identification.

We used the sequence-specific endonucleases EcoRI, SmaI, BamHI, HsuI, and HaeIII as identification tools in following the conjugal transfer of the well-studied R plasmids Sa, R388, RP4, and R6K. Transfers were both intergeneric and intrageneric. Plasmid fingerprints were generated from both single- and combination-enzyme digests. The Sa transconjugants yielded plasmids showing consistent fingerprints for each of the respective endonucleases used, whereas the three other R-plasmid transconjugants showed fingerprint changes.     

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.     

Deletions in the r-determinant mer region of plasmid R100-1 selected for loss of mercury hypersensitivy.

A mutant of plasmid R100-1, which conferred cellular hypersensitivity to Hg2+ because of the insertion of Tn801 (TnA) into the gene determining synthesis of mercuric reductase enzyme, allowed further mutational events to be selected which resulted in either reversion to Hg2+ resistance (characteristic plasmid R100-1) or sensitivity at a level characteristic of plasmidless strains. Restriction endonuclease EcoRI and BamHI analysis showed that reversion to resistance resulted from loss of TnA from the R100-mer:Tn801 plasmid, whereas the change from hypersensitivity to sensitivity to Hg2+ usually resulted from deletion of part or all of Tn801 plus plasmid deoxyribonucleic acid sequences corresponding to the operator-proximal end of the mer operon.     

Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286

The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.     

Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance

The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.     

The stable carriage of two TnA units on a single replicon

Summary Bacterial plasmids which contain a copy of TnA are refractory to the uptake of a second by transposition. However plasmids containing two such copies can be constructed by in vitro recombination techniques. Some plasmids containing two copies of TnA have been obtained by conventional transposition, but in all cases they arose by the virtually simultaneous insertion of both units into a replicon that carried no TnA. All stable plasmids containing two copies of TnA carried the transposons in opposite orientation.     

Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b.

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.     

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.     

Inhibition of TnA translocation by TnA.

Plasmids already containing TnA showed decreased susceptibility to the translocation of a further TnA unit when compared with related plasmids that did not contain TnA. The translocation immunity imposed by TnA is exerted only on the plasmid of which it is part. It is suggested that this desensitization by a translocation unit is a general phenomenon that reduces the mutational effects of translocation.     

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.     

Characterization of small ampicillin resistance plasmids (Rsc) originating from the mutant antibiotic resistance factor R1drd-19B2.

Summary Four plasmids Rsc10–13 ranging in size from 5.1×10⁶ to 13.4×10⁶ Dalton have been isolated from a strain carrying the copy mutant R1drd-19B2 of the antibiotic resistance factor R1. The Rsc plasmids have been cloned by transformation in Escherichia coli C. They determine high level resistance to ampicillin and occur in the cell in multiple copies. Their copy number and stability in the bacterial cell depend on the plasmid and the host strain.Physical maps of these plasmids have been constructed by cleavage with restriction endonucleases HincII, EcoRI, HindIII, BamI and SmaI. The pattern of the cleavage fragments have been compared with the parent plasmid R1drd-19B2 and with a R1 deletion mutant, R1drd-16, which has lost the ampicillin resistance. For Rsc11 and Rsc10 the data indicate, that both plasmids derive from a continuous stretch of the R1drd-19B2 DNA extending from the ampicillin transposon (TnA) to the replication site of the R1 factor. The plasmids Rsc12 and 13 have lost a DNA sequence between TnA and the replication site of R1. They may be formed by translocation of TnA to different autonomously replicating fragments of R1drd-19B2 including the replication origin or by deletion of DNA sequences from Rsc10 and Rsc11.     

Translocation of an ampicillin resistance determinant within an R-factor aggregate inSalmonella panama

The molecular properties of the plasmids of a natural isolate ofSalmonella panama have been studied. This strain, Sp477, harbours 5 different plasmids: the conjugative plasmid pRI477TF (molecular weight 20 megadaltons), the two non-conjugative plasmids, pRI477A and pRI477S, coding for ampicillin and streptomycin plus sulfonamide resistance respectively (molecular weights of both 5.6 megadaltons) and two cryptic plasmids with molecular weights of 1.0 and 2.7, megadaltons respectively. After conjugal transfer toEscherichia coli the ampicillin resistance determinant was frequently found to be integrated into pRI477TF or pRI477S. The translocatable sequence on pRI477A, designated as Tn901, resembles the TnA subclass transposon TnA(1).     

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.     

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.     

Physical and genetic organization of the plasmid R15

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.     

Isolation and genetic analysis of deletion mutants of colicin E1 plasmids carrying a TnA insertion.

Deletions of colicin E1 (colE1) plasmid deoxyribonucleic acid (DNA) carrying the TnA transposon have been isolated. All except two were generated by nuclease digestion of plasmid DNA from its EcoRI-sensitive site. A plasmid containing about 16% of the ColE1 DNA (6.5 X 10(5) daltons) was generated that also contained the part of the TnA transposon conferring ampicillin resistance. The extents of different deletions were determined by analysis of restriction endonuclease fragments generated by the restriction endonucleases HaeII, BamHI, and HincII.     

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.     

Comparative analysis of the structure of incompatibility plasmids N, P and W

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.