The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19

Summary The region of plasmid R1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wildtype R1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.     

Restriction map of the antibiotic resistance plasmid R1drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI.

Summary The conjugative R plasmid R1drd-19, mediating antibiotic resistance to ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm) and sulfonamides (Su) was mapped using the restriction endonucleases BamHI, HindIII, EcoRI and SalI. BamHI generates 5 fragments (A-E) with molecular weights between 46×10⁶ dalton (representing mainly the RTF) and 0.25×10⁶ dalton, and HindIII 8 (A-H) between 42×10⁶ dalton (representing the main part of the RTF) and 0.1×10⁶ dalton. EcoRI recognises 17 sites and produces fragments (A-Q) with molecular weights between 11.7 and 0.1×10⁶ dalton. SalI yields 7 fragments (A-G) of 16.5 to 2.0×10⁶ dalton.A physical map was constructed from fragments obtained by partial digestion of R1drd-19 with one restriction enzyme, by double and triple digestion of the DNA with two or three enzymes with and without isolation of individual bands from preparative gels. In addition the restriction patterns of several mutants of R1drd-19 were compared with it.Evidence is presented which indicates that the derivatives of R1 investigated are generated by extende deletions, namely the copy mutant pKN102 which has lost the Km resistance, R1 drd-16, which has lost all resistances other than Km and the Km^s derivative of R1drd-16, which represents the pure RTF. The map of R1drd-19 is remarkably different from those of R100 and R6-5. Its molecular weight was estimated to be 62.5 Md. The circular fragment order for BamHI is: A-C-B-D-E, for HindIII: A-D-C-B-F-H-E-G, for EcoRI: A-C-K-B-F-J-O-D-H-L-G-P-Q-N-I-E-M-and for SalI A-B-C-D-G-F-E.     

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19

Summary We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kb long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.     

Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.     

Identification of the plasmid R1drd-19 region complementing the mutant phenotype recB- in Escherichia coli K12 strain

Plasmid R1-19 and its copy number mutants markedly increase the recombinational efficiency of a recB- strain of E. coli K12 and its resistance to the lethal action of UV and mitomycin C. These effects are associated with the appearance of a new ATP-dependent exonuclease activity in recB- cells known to be deficient in the ATP-dependent exonuclease V. Using hybrid plasmids carrying different EcoRI fragments of R1-19 (in the pSF124 vector), the gene(s) responsible for effect of R1-19 in recB-cells were localized in the EcoRI-C fragment (8.5 MD) belonging to the RTF portion of R1-19. Expression of the gene(s) in hybrid plasmids depends on the orientation of EcoRI-C fragment in the vector. The copy number of the EcoRI-C fragment was not strictly correlated with the degree of expression of the effects in the recB- mutant.     

A 140 base-pair DNA segment from the kanamycin resistance region of plasmid R1 acts as an origin of replication and promotes site-specific recombination

A 140 base-pair DNA segment situated just upstream of the kanamycin resistance gene of transposon Tn2350, a transposon carried by the plasmid R1, was found to act as an origin of replication and allow autonomous replication of a plasmid composed only of the segment and of the tetracycline resistance gene of pBR322. This segment also promotes site-specific recombination: when cloned in pBR322 it promotes multimer formation in a recA- strain. If two copies are cloned on the same plasmid they promote either deletion or inversion of the intervening region, depending on their orientation relative to each other. DNA gyrase seems to be involved in this process since the inversion rate, in a plasmid carrying sequences in opposite orientations, varies in different nalidixic acid-resistant strains (gyr A mutants) independently isolated.     

R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19.

Copy mutants of the R plasmid R1drd-19 were used to study gene dosage effects in Escherichia coli K-12. The specific activity of β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylylase, as well as ampicillin resistance, increased linearly with the gene dosage up to a level at least tenfold higher than that of the wild-type plasmid. This makes it possible to use ampicillin resistance to determine plasmid copy number and also to select for plasmid copy mutants with defined copy number. Chloramphenicol resistance, despite the increase in enzyme activity, reached a plateau level at a gene dosage less than twice that of the wild-type plasmid, presumably due to the high energy demand on the cells during inactivation of the antibiotic by acetylation with acetyl-coenzyme A. Similarly, resistance to streptomycin plateaued at a gene dosage about three times that of the wild-type plasmid, presumably because of a decreased efficiency of the cells' outer penetration barriers when carrying the R plasmid. The susceptibility of the cells to rifampicin was increased by the presence of plasmid copy mutants.     

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.     

Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19.

Plasmid R1drd-19 is present in a small number of copies per cell of Escherichia coli. The plasmid was reduced in size by in vivo as well as in vitro (cloning) techniques, resulting in a series of plasmid derivatives of different molecular weight. All plasmids isolated contain a small region (about 2 x 10(6) daltons of deoxyribonucleic acid) of the resistance transfer factor part of the plasmid located close to one of the IS1 sequences that separates the resistance transfer factor part from the resistance determinant. All these derivatives were present at the same copy number, retained the incompatibility properties of plasmid R1drd-19, and were stably maintained during cell division. Genes mutated to yield copy mutations also were found to be located in the same region.     

Transfer of kanamycin resistance mediated by plasmid R68.45 in Paracoccus denitrificans.

Plasmid R68.45 mediates the transfer of kanamycin resistance from Pseudomonas aeruginosa to Paracoccus denitrificans. Kanamycin resistance could be transferred from one strain of P. denitrificans to another, thus opening up the possibility of using R68.45 as a sex factor in P. denitrificans.     

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATP-dependent ds DNA exonuclease in recB/R1drd-19 cells. This paper further investigates the suppressive effect of plasmid R1drd-19 on the recB mutation of E. coli. The gene(s) responsible for the effect was localized to the 13.1-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of R1drd-19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of super oligomerization of the tester pACYC184 plasmid in recB/R1drd-19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by lambda Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. coli chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of R1drd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway.     

The kanamycin resistance transposon Tn2680 derived from the R plasmid Rts1 and carried by phage P1Km has flanking 0.8-kb-long direct repeats

Phage P1Km carries within the invertible DNA segment a 5-kb insertion with 0.8-kb terminal direct repeats flanking the kanamycin resistance determinant. The same structure was also found on the R plasmid Rts1, from which the Km resistance segment of P1Km was derived. Obviously, this Km resistance segment translocated as a unit to the P1 genome and it is therefore called Tn2680. Loss of the Km resistance determinant due to recombination between the flanking direct repeats occurs during vegetative growth of P1Km. Amplification of Tn2680 to tandem oligomers is documented and is thought to result from recombination between the flanking direct repeats. The flanking 0.8-kb repeats are different from known IS elements.     

Cloning the origin of transfer region of the resistance plasmid R1

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.     

Intra- and intermolecular recombination of test plasmids in K12 Escherichia coli cells carrying an RTF derivative of the R1drd-19 plasmid

The RTF derivative of the plasmid R1drd-19 was found to stimulate recombination of the tester plasmids in a recB mutant of Escherichia coli K12. The frequency of intramolecular recombination is increased 3.5 and 20-fold, as compared to the one in rec+ and rec- strains, respectively. The frequency of interplasmid recombination is enhanced 4 and 9-fold, respectively. Considerable heterogeneity of the recombination products of the tester plasmid intramolecular recombination in recB-/RTFR1-19 strain has been revealed. It is hypothesized that a "recombinase" encoded by Rldrd-19 plasmid determines a new minor pathway in recB- (Rec P) which differs in activity and, perhaps substrate specificity from the main Rec BCD pathway.     

Isolation and structure of the replicon of the promiscuous plasmid pCU1

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.